FN3 domain-siRNA conjugates and uses thereof

Abstract

The present disclosure relates to compositions, such as siRNA molecules and FN3 domains conjugated thereto, as well as methods of making and using the molecules.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 380,112, filed Oct. 19, 2022, and U.S. Provisional Application No. 63 / 505,898, filed Jun. 2, 2023, each of which is hereby incorporated by reference in its entirety. Technical field

[0003] Embodiments of the present invention relate to siRNA molecules that can be conjugated to fibronectin type III domains (FN3) and methods of making and using such molecules. Background art

[0004] Therapeutic nucleic acids include, for example, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense agents, antagomirs, antimir, microRNA mimics, supermir, U1 adaptors, and aptamers. In the case of siRNAs or miRNAs, these nucleic acids can down - regulate the intracellular levels of specific proteins via a process called RNA interference (RNAi). The therapeutic applications of RNAi are extremely broad because siRNA and miRNA constructs can be synthesized with any nucleotide sequence against the transcript of any target protein. To date, siRNA constructs have shown the ability to specifically down - regulate target proteins in in vitro and in vivo models. Additionally, siRNA constructs are currently being evaluated in clinical studies and have been approved for a variety of diseases.

[0005] However, siRNA constructs currently face two problems: first, their susceptibility to nuclease digestion in plasma, and second, their limited ability to enter intracellular compartments and bind to RISC (RNA - induced silencing complex) when administered systemically as free siRNAs or miRNAs. Certain delivery systems, such as lipid nanoparticles formed from cationic lipids and other lipid components (e.g., cholesterol and PEG lipids), carbohydrates (e.g., GalNAc trimers), have been used to facilitate cellular uptake of oligonucleotides. However, these delivery systems have not been shown to successfully deliver siRNAs efficiently and effectively to their intended targets in tissues other than the liver.

[0006] CD40 and its ligand CD40L (or CD154) are transmembrane proteins expressed by immune cells, including B cells, T cells, and dendritic cells. CD40 serves to enhance the immune response by stimulating the activation and maturation of T cells as well as B cells. In autoimmune diseases, the role of CD40 in immune system activation also includes the production of autoantibodies. For example, studies have shown that CD40 plays a role in rheumatoid arthritis, autoimmune thyroid diseases, type I diabetes, neuroinflammatory diseases (such as multiple sclerosis), psoriasis, inflammatory bowel disease, systemic lupus erythematosus, and lupus nephritis (see, e.g., Zheng et al., Arthritis Res. & Therapy, 2010, 12:R13; Peters et al., Semin Immunol., 2009, 21(5):293-300; and Ripoll et al., PLoS One, 2013, 8(6):e65068).

[0007] There is a need for compositions and methods for delivering therapeutic nucleic acids, such as small interfering RNA (siRNA), to a predetermined cellular target to downregulate the production and expression of CD40 in a subject having an autoimmune disease. Further, there is a need for a method of having an FN3 domain that specifically binds CD71 with optimized clinical use characteristics, and novel therapeutic agents that are capable of entering cells via receptor-mediated CD71 internalization using such molecules. Embodiments of the present invention meet these needs and others. SUMMARY OF THE INVENTION

[0008] The present disclosure provides compositions comprising siRNA molecules, which molecules comprise a sense strand and an antisense strand, such as those provided herein. In some embodiments, the siRNA molecule targets the CD40 gene. In some embodiments, the siRNA further comprises a linker covalently linked to the sense or antisense strand of the siRNA. In some embodiments, the linker is linked to the 5' or 3' end of the sense or antisense strand. In some embodiments, the siRNA molecule further comprises a vinyl phosphonate modification on the sense or antisense strand. In some embodiments, the vinyl phosphonate modification is located at the 5' or 3' end of the sense or antisense strand. In some embodiments, the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1890, 1893, 1941, 1942, 1944, 46-178, 312-331, 1850, 1851, 1891, 1892, 1894-1928, 1932-1940, 1943, 1945-1959, 2298, 2302, 2304, 352-356, 673-805, 939-958, 2070, 2071, 2110-2148, 2152-2179, 2300, 2306, and 2308. In some embodiments, the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2290, 2293, 2051, 2052, 2054, 179-311, 332-351, 1960, 1961, 2000-2038, 2042-2050, 2053, 2055-2069, 2291, 2292, 2294-2297, 2299, 2303, 2305, 356-359, 806-938, 959-978, 2180, 2181, 2220-2258, 2262-2289, 2301, 2307, and 2309. In some embodiments, the siRNA molecule comprises a paired siRNA shown in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B.

[0009] In some embodiments, the composition further comprises one or more FN3 domains conjugated to the siRNA molecule. In some embodiments, the one or more FN3 domains comprise an FN3 domain that binds CD71. In some embodiments, the FN3 domain comprises an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or completely identical to any one of the sequences selected from SEQ ID NO: 570, 672, 1848, 1773, 1849, 1767, 360 - 569, 571 - 644, 663 - 67, 1395 - 1772, 1774 - 1766, and 1768 - 1847.

[0010] In some embodiments, the one or more FN3 domains comprise at least two FN3 domains linked by a peptide linker. In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 645 - 661.

[0011] The present invention also provides a composition having the following formula:

[0012] (X1) n -(X2) q -(X3) y -L-X4;

[0013] C-(X1) n -(X2) q -L-X4-(X3) y ;

[0014] (X1) n -(X2) q -L-X4-(X3) y -C;

[0015] C-(X1) n -(X2) q -L-X4-L-(X3) y ;

[0016] or (X1) n -(X2) q -L-X4-L-(X3) y -C,

[0017] Wherein: X1 is the first FN3 domain; X2 is the second FN3 domain; X3 is the third FN3 domain or a half-life extending molecule; L is a linker; and X4 is a nucleic acid molecule, such as an siRNA targeting CD40, such as the nucleic acid molecules provided herein. C is a polymer, such as PEG, albumin-binding protein or an aliphatic chain that binds to serum proteins, wherein n, q and y are each independently 0 or 1. In some embodiments, X1, X2 and X3 bind to the same or different target proteins.

[0018] The present invention also provides a composition having the formula A1-B1, wherein A1 has the formula (C) n -(L1) t -X s and B1 has the formula X AS -(L2) q -(F1) y , or A1 has the formula (F1) n -(L1) t -X s and B1 has the formula X AS -(L2) q -(C) y , wherein:

[0019] C is a polymer, such as PEG, albumin-binding protein or an aliphatic chain that binds to serum proteins;

[0020] L1 and L2 are each independently a linker;

[0021] X S is the 5' to 3' oligonucleotide sense strand of a double-stranded siRNA molecule;

[0022] X AS is the 3' to 5' oligonucleotide antisense strand of a double-stranded siRNA molecule;

[0023] F1 is a polypeptide comprising at least one FN3 domain;

[0024] wherein n, t, q and y are each independently 0 or 1;

[0025] wherein X S and X AS form a double-stranded oligonucleotide molecule to form a composition / complex targeting CD40.

[0026] ​​​​In some embodiments, provided herein is a method of treating an immune disease in a subject in need thereof, the method comprising administering to the subject a composition, such as any composition provided herein. In some embodiments, provided herein is a method of reducing the mRNA expression of a target gene in a cell (such as an immune cell), the method comprising contacting the immune cell with a composition such as any composition provided herein. In some embodiments, provided herein is a method of delivering an siRNA molecule to a cell (such as an immune cell) of a subject, the method comprising administering to the subject a pharmaceutical composition comprising any composition provided herein. In some embodiments, provided herein is a method of delivering an siRNA molecule targeting CD40 to an immune cell expressing CD71 of a subject, the method comprising administering to the subject a pharmaceutical composition comprising any composition provided herein, wherein the siRNA molecule downregulates the mRNA expression of CD40 in the immune cell expressing CD71.

[0027] Further provided herein is a method of reducing one or more serum cytokines, the method comprising administering an siRNA molecule targeting CD40 to one or more immune cells expressing CD71. In some embodiments, the one or more serum cytokines comprise IFN-γ, IL-6, TNF-α, IL-12, IP-10, RANTES, or any combination thereof. In some embodiments, the one or more immune cells expressing CD71 comprise B cells, T cells, or a combination thereof.

[0028] Further provided herein is a method of selectively reducing a population of immune cells expressing CD71, the method comprising administering an siRNA molecule targeting CD40 to the population of immune cells expressing CD71. In some embodiments, the population of immune cells expressing CD71 comprises B cells, T cells, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flow chart depicting the steps for in silico screening for CD40 siRNA and evaluating properties.

[0030] Figure 2 Depicting Raji cells ( Figure 2 , Panel A) and A20 cells ( Figure 2 , Panel B) titration curves of exemplary CD40 siRNAs.

[0031] Figure 3ADepict the relative CD40 mRNA expression in vitro of donor human dendritic cells that have been activated and exposed to CD40 ligand, with or without treatment with an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40.

[0032] Figure 3B Depict the in vitro IL-12 production of donor human dendritic cells that are activated or not activated, exposed or not exposed to CD40 ligand, and treated or not treated with an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40.

[0033] Figure 4 Depict the relative CD40 mRNA expression in vitro of donor human dendritic cells that have been activated and treated with increasing concentrations of an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40. The CD40 mRNA expression of the treated cells is normalized to the mRNA expression of activated, untreated dendritic cells. As the concentration of the conjugate (in nM) increases, the relative CD40 mRNA expression decreases for all donors.

[0034] Figure 5 Depict the relative CD40 protein expression in vitro over time of donor human dendritic cells that have been activated and treated with an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40, or activated and untreated ("activated only"). The CD40 protein expression of the treated cells is normalized to the protein expression of activated, untreated dendritic cells.

[0035] Figure 6 Depict the in vitro cytokine production of donor dendritic cells that have been activated and treated with an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40, donor dendritic cells that have been activated and treated with a negative control, and donor dendritic cells that have been activated and untreated.

[0036] Figure 7 Depict the in vivo serum cytokine levels of mice that have been activated and treated with an exemplary FN3 domain conjugated to CD71 and an siRNA conjugate targeting CD40, mice that have been activated and treated with a negative control, mice that have been activated and treated only with the FN3 domain conjugated to CD71, mice that have been activated and treated with a vehicle, and naive mice that have neither been activated nor treated.

[0037] Figure 8Depict the in vivo serum cytokine levels in mice induced with EAE (an animal disease model), activated, and treated with an exemplary FN3 domain that binds CD71 and a siRNA conjugate that targets CD40. Further depict EAE mice that are activated and treated with a negative control, EAE mice that are activated and treated only with the FN3 domain that binds CD71, EAE mice that are activated and treated with a vehicle, and healthy naive mice that are neither activated nor treated.

[0038] Figure 9 Depict the in vivo frequencies of B cells in draining lymph node tissue and spinal cord tissue collected from mice induced with EAE (an animal disease model), activated, and treated with an exemplary FN3 domain that binds CD71 and a siRNA conjugate that targets CD40. Further depict EAE mice that are activated and treated with a positive control, EAE mice that are activated and treated with a vehicle, and healthy naive mice that are neither activated nor treated.

[0039] Figure 10 Depict the in vivo frequencies of dendritic cells, CD8 T cells, and CD4 T cells in spinal cord tissue collected from mice induced with EAE (an animal disease model), activated, and treated with an exemplary FN3 domain that binds CD71 and a siRNA conjugate that targets CD40. Further depict EAE mice that are activated and treated with a positive control and EAE mice that are activated and treated with a vehicle.

[0040] Figure 11 Depict the in vivo frequencies of lymphocytes, monocytes, and macrophages in spinal cord tissue collected from mice induced with EAE (an animal disease model), activated, and treated with an exemplary FN3 domain that binds CD71 and a siRNA conjugate that targets CD40. Further depict EAE mice that are activated and treated with a positive control and EAE mice that are activated and treated with a vehicle. Detailed Description

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0042] "Fibronectin type III domain" or "FN3 domain" refers to a polypeptide sequence that occurs frequently in proteins including fibronectin, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-naturally synthesized FN3 domains such as those described in U.S. Patent No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3rd FN3 domain of tenascin (TN3) or the 10th FN3 domain of fibronectin (FN10). As used throughout, "Centyrin" also refers to an FN3 domain. In addition, the FN3 domains described herein are not antibodies because they do not have the structure of a variable heavy chain (V H ) and / or a light chain (V L ).

[0043] "Autoimmune disease" refers to a disease condition and state in which an individual's immune response targets the individual's own components, resulting in an undesired and often debilitating disorder. As used herein, "autoimmune disease" is also intended to include autoimmune disorders, syndromes, etc. Autoimmune diseases include, but are not limited to, Addison's disease, allergy, allergic rhinitis, ankylosing spondylitis, asthma, atherosclerosis, autoimmune ear diseases, autoimmune eye diseases, autoimmune atrophic gastritis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune parotitis, autoimmune uveitis, celiac disease, primary biliary cirrhosis, benign lymphocytic vasculitis, COPD, colitis, coronary heart disease, Crohn's disease, diabetes (type I), depression, diabetes (including type 1 and / or type 2 diabetes), epididymitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD), immune response to recombinant drugs (such as factor VII for hemophilia), juvenile idiopathic arthritis, systemic lupus erythematosus, lupus nephritis, male infertility, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neoplasm, osteoarthritis, pain, primary myxedema, pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, sympathetic ophthalmia, T-cell lymphoma, T-cell acute lymphoblastic leukemia, testicular angioimmunoblastic T-cell lymphoma, thyroiditis, transplant rejection, ulcerative colitis, autoimmune uveitis, and vasculitis. Autoimmune diseases include, but are not limited to, disorders in which the affected tissue is the primary target and in some cases the secondary target. Such disorders include, but are not limited to, AIDS, atopic allergy, bronchial asthma, eczema, leprosy, schizophrenia, hereditary depression, tissue and organ transplantation, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus' phenomenon, systemic anaphylaxis, and alcohol and drug addiction.

[0044] "Capturing agent" refers to a substance that binds to a specific type of cell and is capable of separating the said cell from other cells. Exemplary capturing agents are magnetic beads, ferrofluids, encapsulating reagents, molecules that bind to specific cell types, etc.

[0045] "Sample" means a collection of like bodily fluids, cells or tissues isolated from a subject, as well as bodily fluids, cells or tissues present in a subject. Exemplary samples are tissue biopsies, fine needle aspirates, surgically removed tissues, organ cultures, cell cultures and biological fluids such as blood, serum and serous fluid, plasma, lymph fluid, urine, saliva, cyst fluid, teardrops, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid and fluids of other body cavities, fluids collected by bronchoalveolar lavage, synovial fluid, fluid solutions in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media and lavage fluids, etc.

[0046] "Substituted", "being substituted", "mutated" or "having been mutated" means changing, deleting or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to produce a variant of said sequence.

[0047] "Variant" means a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications (such as substitutions, insertions or deletions).

[0048] "Specifically binds" or "specific binding" means the ability of an FN3 domain to bind to its target (such as CD71), the dissociation constant (K D ) of which is about 1x10 -6 M or less, such as about 1x10 -7 M or less, about 1x10 -8 M or less, about 1x10 -9 M or less, about 1x10 -10 M or less, about 1x10 -11 M or less, about 1x10 -12 M or less, or about 1x10 -13 M or less. Alternatively, "specific binding" means that in a standard solution ELISA assay, the ability of an FN3 domain to bind to its target (such as CD71) is at least 5-fold higher than that of a negative control. Specific binding can also be demonstrated using a proteome array as described herein. In some embodiments, the negative control is an FN3 domain that does not bind CD71. In some embodiments, an FN3 domain that specifically binds CD71 may be cross-reactive with other related antigens, such as with the same predetermined antigen (homologue) from other species such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

[0049] "Library" refers to a collection of variants. A library can consist of polypeptide or polynucleotide variants.

[0050] "Stability" refers to the ability of a molecule to maintain its folded state under physiological conditions such that it retains at least one normal functional activity, such as the ability to bind to a predetermined antigen, such as CD71.

[0051] "CD71" refers to the human CD71 protein having the amino acid sequence of SEQ ID NO: 3 or 4. In some embodiments, SEQ ID NO: 3 is the full-length human CD71 protein. In some embodiments, SEQ ID NO: 4 is the extracellular domain of human CD71.

[0052] "Tencon" refers to a synthetic fibronectin type III (FN3) domain having the following consensus sequence:

[0053]

[0054] and is described in U.S. Patent Publication No. 2010 / 0216708.

[0055] "Immune cell" refers to a cell classified in the immune system as a lymphocyte (T cell, B cell, and NK cell), neutrophil, or monocyte / macrophage. Immune cells also include dendritic cells. "Dendritic cell" refers to a type of antigen-presenting cell (APC) that plays an important role in the adaptive immune system. The main function of dendritic cells is to present antigens to T lymphocytes and secrete cytokines that can further directly or indirectly regulate the immune response. Dendritic cells have the ability to induce a primary immune response in naive T lymphocytes that are inactive or resting.

[0056] "Vector" refers to a polynucleotide capable of replicating within a biological system or being movable between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a polyadenylation signal, or a selectable marker to facilitate the duplication or maintenance of these polynucleotides within a biological system. Examples of such biological systems can include cells, viruses, animals, plants, and reconstituted biological systems utilizing biological components capable of replicating the vector. The polynucleotide constituting the vector can be a DNA or RNA molecule or a hybrid of these substances.

[0057] "Expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0058] "Polynucleotide" refers to a synthetic molecule containing a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical means. cDNA is a typical example of a polynucleotide.

[0059] "Polypeptide" or "protein" refers to a molecule that contains at least two amino acid residues joined by peptide bonds to form a polypeptide. A small polypeptide of less than about 50 amino acids may be referred to as a "peptide".

[0060] "Valency" refers to the presence of a specified number of antigen-specific binding sites in a molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of one, two, four and six antigen-specific binding sites in a molecule, respectively.

[0061] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise specified, the terms "patient" or "subject" may be used interchangeably.

[0062] "Isolated" refers to a homogeneous population of molecules (such as synthetic polynucleotides or polypeptides such as FN3 domains) that have been substantially separated and / or purified from other components in the system (such as a recombinant cell) that produces the molecules, and proteins that have undergone at least one purification or isolation step. "Isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular materials and / or chemicals, and encompasses FN3 domains isolated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0063] "Migration", when used to refer to the movement of a cell, means the movement of a cell from one location to another. For example, the movement of a cell (such as a white blood cell or immune cell) from a blood vessel to a tissue can be referred to as migration from the blood vessel to the tissue. Migration can also include the process of "margination", which refers to the movement of a cell from the interior of a blood vessel towards the vessel wall. Migration can also include the adhesion of a cell to the vessel wall, and the migration across the vessel wall into the tissue.

[0064] Composition

[0065] In some embodiments, a composition is provided that comprises a polypeptide linked to an oligonucleotide molecule, such as a polypeptide comprising an FN3 domain. The oligonucleotide molecule can be, for example, an siRNA molecule. In some embodiments, the FN3 domain is an FN3 domain that binds CD71 as provided herein. In some embodiments, the oligonucleotide is a CD40 siRNA that binds to CD40 RNA (such as the mRNA provided herein). In some embodiments, the composition further comprises a polymer as provided herein.

[0066] In some embodiments, the siRNA molecule is a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand (lagging strand) and an antisense strand (guide strand). In some embodiments, the length of each strand of the dsRNA agent can range from 12 to 40 nucleotides. For example, the length of each strand can be 14 - 40 nucleotides, 17 - 37 nucleotides, 25 - 37 nucleotides, 27 - 30 nucleotides, 17 - 23 nucleotides, 17 - 21 nucleotides, 17 - 19 nucleotides, 19 - 25 nucleotides, 19 - 23 nucleotides, 19 - 21 nucleotides, 21 - 25 nucleotides, or 21 - 23 nucleotides.

[0067] In some embodiments, the sense strand and the antisense strand generally form a double helix dsRNA. The length of the double helix region of the dsRNA agent can be 12 - 40 nucleotide pairs. For example, the length of the double helix region can be 14 - 40 nucleotide pairs, 17 - 30 nucleotide pairs, 25 - 35 nucleotides, 27 - 35 nucleotide pairs, 17 - 23 nucleotide pairs, 17 - 21 nucleotide pairs, 17 - 19 nucleotide pairs, 19 - 25 nucleotide pairs, 19 - 23 nucleotide pairs, 19 - 21 nucleotide pairs, 21 - 25 nucleotide pairs, or 21 - 23 nucleotide pairs. In another example, the length of the double helix region is selected from 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotide pairs.

[0068] In some embodiments, the dsRNA comprises one or more overhang regions and / or capping groups of the dsRNA agent at the 3' end or 5' end or both ends of one strand. The length of the overhang can be 1 - 10 nucleotides, 1 - 6 nucleotides, such as 2 - 6 nucleotides, 1 - 5 nucleotides, 2 - 5 nucleotides, 1 - 4 nucleotides, 2 - 4 nucleotides, 1 - 3 nucleotides, 2 - 3 nucleotides, or 1 - 2 nucleotides. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence. The first strand and the second strand can also be joined, for example, by additional bases to form a hairpin, or joined by other non-base linkers.

[0069] In some embodiments, the nucleotides in the overhang region of the dsRNA agent can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modifications such as 2'-F, 2'-O-methyl, 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), 2'-O-(2-methoxyethyl), and any combination thereof. For example, TT(UU) can be the overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence.

[0070] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the dsRNA agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate, dithiophosphate, phosphonate, aminophosphate, or methylsulfonamidophosphate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

[0071] The dsRNA agent can contain only a single overhang, which can enhance the interfering activity of the dsRNA without affecting its overall stability. For example, the single-stranded overhang is located at the 3'-end of the sense strand or the 3'-end of the antisense strand. The dsRNA can also have blunt ends, located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3'-end and a blunt end at the 5'-end. Although not bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand facilitate the loading of the guide strand into the RNA-induced silencing complex (RISC). For example, the length of the single overhang contains at least two, three, four, five, six, seven, eight, nine, or ten nucleotides.

[0072] In some embodiments, the dsRNA agent can also have two blunt ends at both ends of the dsRNA duplex.

[0073] In some embodiments, each nucleotide in the sense strand and antisense strand of the dsRNA agent can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or both of the non-linking phosphate oxygens and / or one or more alterations of one or more of the linking phosphate oxygens; alterations of the ribose moiety, such as alterations of the 2'-hydroxyl on the ribose; bulk replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of the naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0074] In some embodiments, all or some of the bases in the 3' or 5' overhangs can be modified, for example, with the modifications described herein. Modifications can include, for example, using modifications at the 2'-position of the ribose with modifications known in the art, such as using deoxyribonucleotides modified with 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl (2'-OMe) in place of the ribose of the nucleobase, and modifications of the phosphate group, such as phosphorothioate, dithiophosphate, phosphonate, aminophosphate, or methylsulfonamidophosphate modifications. The overhangs need not be homologous to the target sequence.

[0075] In some embodiments, each residue of the sense and antisense strands is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro.

[0076] In some embodiments, there are typically at least two different modifications present on the sense and antisense strands. The two modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, or other modifications.

[0077] In one embodiment, the sense and antisense strands each contain two nucleotides modified in different ways selected from 2'-fluoro, 2'-O-methyl, or 2'-deoxy.

[0078] The dsRNA agent can also contain at least one phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkage. The phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkage modification can occur on any nucleotide at any position in the sense or antisense strand or both strands. For example, the internucleotide linkage modification can occur on each nucleotide of the sense and / or antisense strand; each internucleotide linkage modification can occur in an alternating pattern on the sense or antisense strand; or the sense or antisense strand contains two internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand can be shifted relative to the alternating pattern of internucleotide linkage modifications on the antisense strand.

[0079] In some embodiments, the dsRNA agent comprises phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkage modifications in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkage therebetween. Internucleotide linkage modifications can also be made to link the overhang nucleotides to the terminal paired nucleotides within the double helix region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked via phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkages, and optionally, additional phosphorothioate, dithiophosphate, phosphonate, aminophosphate, methylsulfonamidophosphate, or methylphosphonate internucleotide linkages may be present to link the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. In some embodiments, these terminal three nucleotides can be located at the 3' end of the antisense strand.

[0080] In some embodiments, the dsRNA composition is linked by a modified base or nucleoside analogue as described in U.S. Patent No. 7,427,672, which is incorporated herein by reference. In some embodiments, the modified base or nucleoside analogue is referred to as a linker or L in the formulas described herein.

[0081] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts:

[0082]

[0083] wherein the base represents an aromatic heterocyclic group or an aromatic hydrocarbon ring group optionally having substituents, R1 and R2 are the same or different and each represents a hydrogen atom, a protecting group for the hydroxyl group used in nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or --P(R4)R5, wherein R4 and R5 are the same or different and each represents a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted by an alkyl group having 1 to 5 carbon atoms, and X represents OMe or F.

[0084] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl group, a lower alkoxy group, a halogen atom or a cyano group, or a silyl group.

[0085] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a mesyl group, a tosyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group or a tert-butyldiphenylsilyl group.

[0086] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl group, a lower alkoxy group, a halogen atom or a cyano group, a silyl group, an aminophosphite group, a phosphonyl group, a phosphate group or a phosphate group protected with a protecting group for nucleic acid synthesis.

[0087] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a mesyl group, a tosyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, --P(OC2H4CN)(N(i-Pr)2), --P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl phosphate group or a 4-chlorophenyl phosphate group.

[0088] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein the base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following α groups: a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms and a halogen atom.

[0089] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula I and its salts, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0090] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts:

[0091]

[0092] Wherein the base represents an optionally substituted aromatic heterocyclic group or aromatic hydrocarbon ring group, R1 and R2 are the same or different and each represents a hydrogen atom, a protecting group for the hydroxyl group used in nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or --P(R4)R5, wherein R4 and R5 are the same or different and each represents a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted by an alkyl group having 1 to 5 carbon atoms, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a functional molecule unit substituent, and m represents an integer from 0 to 2, and n represents an integer from 0 to 3. In some embodiments, both m and n are 0.

[0093] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl group, a lower alkoxy group, a halogen, or a cyano group, or a silyl group.

[0094] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a triphenylmethyl group, a dimethoxytriphenylmethyl group, a monomethoxytriphenylmethyl group, or a tert-butyldiphenylsilyl group.

[0095] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups having an aryl ring substituted with a lower alkyl group, a lower alkoxy group, a halogen, or a cyano group, a silyl group, an amino phosphite group, a phosphonyl group, a phosphate group, or a phosphate group protected by a protecting group for nucleic acid synthesis.

[0096] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, --P(OC2H4CN)(N(i-Pr)2), --P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl phosphate group or a 4-chlorophenyl phosphate group.

[0097] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein R3 is a hydrogen atom, phenoxyacetyl, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with one to three aryl groups, a lower aliphatic or aromatic sulfonyl group (such as mesyl or tosyl), an aliphatic acyl group having 1 to 5 carbon atoms (such as acetyl) or an aromatic acyl group (such as benzoyl).

[0098] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid nicking active functional group, or an intracellular or nuclear translocation signal peptide.

[0099] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein the base is a purin-9-yl, 2-oxopyrimidin-1-yl, or a purin-9-yl or 2-oxopyrimidin-1-yl having a substituent selected from the following α groups: hydroxyl, hydroxyl protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, mercapto, mercapto protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, amino, amino protected with a protecting group for nucleic acid synthesis, amino substituted with an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms and a halogen atom.

[0100] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0101] In some embodiments, the modified base or nucleoside analogue has the structure shown in Chemical Formula IB and its salts, wherein m is 0 and n is 1.

[0102] In some embodiments, the modified base or nucleoside analogue is a DNA oligonucleotide or RNA oligonucleotide analogue that contains one or both or more of the unit structures of one or more types of nucleoside analogues having the structure shown in Chemical Formula II, or a pharmaceutically acceptable salt thereof, provided that the form of linkage between individual nucleosides in the oligonucleotide analogue may contain, in addition to the same phosphodiester bond [--OP(O2 - )O--] as in natural nucleic acids, one or two or more phosphorothioate bonds [--OP(O)(S - )O--], dithiophosphonate bonds [--O2PS2--], phosphonate bonds [--PO(OH)2--], aminophosphonate bonds [--O═P(OH)2--], or methylsulfonamidophosphonate bonds [--OP(O)(N)(SO2)(CH3)O--], and if it contains two or more of one or more types of these structures, the bases between these structures may be the same or different:

[0103]

[0104] wherein the base represents an optionally substituted aromatic heterocyclic group or aromatic hydrocarbon ring group, and X represents OMe or F.

[0105] In some embodiments, the oligonucleotide analogue or its pharmaceutically acceptable salt has the structure shown in Chemical Formula II, wherein the base is a purin-9-yl, 2-oxopyrimidin-1-yl, or purin-9-yl or 2-oxopyrimidin-1-yl having a substituent selected from the following α groups: hydroxyl, hydroxyl protected with a protecting group for nucleic acid synthesis, alkoxy having 1 to 5 carbon atoms, mercapto, mercapto protected with a protecting group for nucleic acid synthesis, alkylthio having 1 to 5 carbon atoms, amino, amino protected with a protecting group for nucleic acid synthesis, amino substituted with an alkyl having 1 to 5 carbon atoms, alkyl having 1 to 5 carbon atoms, and halogen atom.

[0106] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula II, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0107] In some embodiments, the modified base or nucleoside analog is a DNA oligonucleotide or an RNA oligonucleotide analog that contains one or both or more of the unit structures of one or more types of nucleoside analogs having a structure as shown in Chemical Formula IIB, or a pharmaceutically acceptable salt thereof, provided that the form of the linkage between individual nucleosides in the oligonucleotide analog may contain, in addition to the same phosphodiester bond [--OP(O2 - )O--] as in natural nucleic acids, one or two or more phosphorothioate bonds [--OP(O)(S -)O--], dithiophosphate bond [--O2PS2--], phosphonate bond [--PO(OH)2--], aminophosphonate bond [--O=P(OH)2--] or mesylaminophosphonate bond [--OP(O)(N)(SO2)(CH3)O--], and if it contains two or more of one or more types of these structures, the bases between these structures may be the same or different:

[0108]

[0109] wherein the base represents an optionally substituted aromatic heterocyclic group or aromatic hydrocarbon ring group, R3 represents a hydrogen atom, alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, sulfonyl, silyl or functional molecular unit substituent, and m represents an integer from 0 to 2, and n represents an integer from 0 to 3. In some embodiments, both m and n are 0.

[0110] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Formula IIB, wherein R1 is a hydrogen atom, aliphatic acyl, aromatic acyl, aliphatic or aromatic sulfonyl, methyl substituted with one to three aryl groups, methyl substituted with one to three aryl groups having an aryl ring substituted with lower alkyl, lower alkoxy, halogen or cyano, or silyl.

[0111] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Formula IIB, wherein R1 is a hydrogen atom, acetyl, benzoyl, mesyl, tosyl, benzyl, p-methoxybenzyl, trityl, dimethoxytrityl, monomethoxytrityl or tert-butyldiphenylsilyl.

[0112] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Formula IIB, wherein R2 is a hydrogen atom, aliphatic acyl, aromatic acyl, aliphatic or aromatic sulfonyl, methyl substituted with one to three aryl groups, methyl substituted with one to three aryl groups having an aryl ring substituted with lower alkyl, lower alkoxy, halogen or cyano, silyl, aminophosphite group, phosphonyl, phosphate group or phosphate group protected with a protecting group for nucleic acid synthesis.

[0113] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein R2 is a hydrogen atom, acetyl, benzoyl, benzyl, p-methoxybenzyl, mesyl, tosyl, tert-butyldiphenylsilyl, --P(OC2H4CN)(N(i-Pr)2), --P(OCH3)(N(i-Pr)2), phosphonyl, or 2-chlorophenyl phosphate or 4-chlorophenyl phosphate.

[0114] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein R3 is a hydrogen atom, phenoxyacetyl, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted with one to three aryl groups, a lower aliphatic or aromatic sulfonyl group (such as mesyl or tosyl), an aliphatic acyl group having 1 to 5 carbon atoms (such as acetyl) or an aromatic acyl group (such as benzoyl).

[0115] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein the functional molecular unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid nicking active functional group, or an intracellular or nuclear translocation signal peptide.

[0116] In some embodiments, the oligonucleotide analogue or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein the base is purin-9-yl, 2-oxopyrimidin-1-yl, or purin-9-yl or 2-oxopyrimidin-1-yl having a substituent selected from the following α groups: hydroxy, hydroxy protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, mercapto, mercapto protected with a protecting group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, amino, amino protected with a protecting group for nucleic acid synthesis, amino substituted with an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms and a halogen atom.

[0117] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein the base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guanyl), 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracilyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl) or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having an amino group protected with a protecting group for nucleic acid synthesis.

[0118] In some embodiments, the oligonucleotide analog or a pharmaceutically acceptable salt thereof has a structure as shown in Chemical Formula IIB, wherein m is 0 and n is 1.

[0119] In some embodiments, the dsRNA agent comprises a mismatch with the target, a mismatch within the double helix, or a combination thereof. The mismatch can occur in the overhang region or the double helix region. Base pairs can be ranked based on their tendency to promote dissociation or strand separation (e.g., based on the free energy of association or dissociation of specific pairings, and the simplest approach is to examine pairings on a single-pair basis, but next-nearest neighbor or similar analyses can also be used). In terms of promoting dissociation: A:U is superior to G:C; G:U is superior to G:C; and I:C is superior to G:C (I = inosine). Mismatches, such as non-canonical pairings or pairings other than canonical pairings (as described elsewhere herein), are superior to canonical (A:T, A:U, G:C) pairings; and pairings including universal bases are superior to canonical pairings.

[0120] In some embodiments, the dsRNA agent can comprise a phosphorus-containing group at the 5'-end of the sense strand or the antisense strand. The 5'-end phosphorus-containing group can be a 5'-end phosphate (5'-P), 5'-end phosphorothioate (5'-PS), 5'-end bisphosphorothioate (5'-PS2), 5'-end vinylphosphonate (5'-VP), 5'-end methylphosphonate (MePhos), 5'-end methanesulfonamidophosphonate (5'MsPA), or 5'-deoxy-5'-C-propionyl. When the 5'-end phosphorus-containing group is 5'-end vinylphosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer, such as a trans vinyl phosphate or a cis vinyl phosphate, or a mixture thereof. Representative structures of these modifications can be seen, for example, in U.S. Patent No. 10,233,448, which is hereby incorporated by reference in its entirety.

[0121] In some embodiments, a nucleotide analogue or a synthetic nucleobase comprises a nucleic acid having a modification at the 2'-hydroxy group of the ribose moiety. In some cases, the modification includes H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, straight-chain and branched-chain C1-C 10 chain lengths. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazine, or hydroxyamino) group, an isocyanate group or a cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further comprises additional heteroatoms, such as O, S, N, Se, and each of these heteroatoms can be further substituted by an alkyl as described above. In some cases, the carbon of the heterocyclic group is substituted by nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidinyl.

[0122] In some cases, the modification at the 2'-hydroxy group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. Exemplary chemical structures of the 2'-O-methyl modification of adenosine molecules and the 2'-O-methoxyethyl modification of uridine are depicted below.

[0123]

[0124] In some cases, the modification at the 2'-hydroxy group is a 2'-O-aminopropyl modification, where an extended amine group containing a propyl linker binds the amine group to the 2'-oxygen. In some cases, this modification neutralizes the overall negative charge derived from the phosphate esters of the oligonucleotide molecule by introducing one positive charge from the amine group through each sugar, thereby improving the cellular uptake properties due to its zwitterionic nature. Exemplary chemical structures of 2'-O-aminopropyl nucleoside phosphoramidites are depicted below.

[0125]

[0126] 2'-O-aminopropyl nucleoside phosphoramidite

[0127] In some cases, the modification at the 2'-hydroxy group is a locked or bridged ribose modification (such as locked nucleic acid or LNA), where the oxygen molecule bound at the 2'-carbon is linked to the 4'-carbon through a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. An exemplary illustration of the chemical structure of LNA is depicted below. The illustration shown on the left highlights the chemical connectivity of the LNA monomer. The illustration shown on the right highlights the locked C3'-endo (3E) conformation of the furanose ring of the LNA monomer.

[0128]

[0129] In some cases, the modification at the 2'-hydroxy group includes ethylene nucleic acid (ENA), such as 2'-4'-ethylene-bridged nucleic acid, which locks the sugar conformation into a C3'-endo sugar pucker conformation. ENA is part of a class of bridged nucleic acid modified nucleic acids that also includes LNA. Exemplary chemical structures of ENA and bridged nucleic acids are depicted below.

[0130]

[0131] In some embodiments, additional modifications at the 2'-hydroxy group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA).

[0132] In some embodiments, the nucleotide analogs include modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides having a modification at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 5-methylaminoethyluridine, 5-methoxyuridine, deazapurines (such as 7-deaza-adenosine), 6-azauridine, 6-azacytidine, 6-azathymidine, 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, Q nucleoside, archaeosine, naphthyl and substituted naphthyl, any O-alkylated purines and pyrimidines, and N-alkylated purines and pyrimidines (such as N6-methyladenosine), 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl (such as aminophenol or 2,4,6-trimethoxybenzene), modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides modified with respect to the sugar moiety and nucleotides having a non-ribosyl sugar or an analogue thereof. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thiothreose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes nucleotides known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0133] In some embodiments, the nucleotide analogs further comprise a morpholino group, peptide nucleic acid (PNA), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-aminophosphonate, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. A morpholino group or a dimethylaminophosphonate morpholino oligomer (PMO) comprises a synthetic molecule whose structure mimics the structure of natural nucleic acids by deviating from the normal sugar and phosphate structures. In some cases, the five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, ribose monomers are linked by dimethylaminophosphonate groups rather than phosphate groups. In such cases, the backbone modification removes all positive and negative charges, enabling the morpholino neutral molecules to cross cell membranes without the assistance of, for example, the cell delivery agents used for charged oligonucleotides.

[0134]

[0135] In some embodiments, peptide nucleic acid (PNA) does not contain a sugar ring or a phosphoester linkage, and the bases are linked and properly spaced by an oligoglycine-like molecule, thus eliminating backbone charges.

[0136]

[0137] In some embodiments, one or more modifications optionally occur at internucleotide linkages. In some cases, modified internucleotide linkages include, but are not limited to, phosphorothioates, methylsulfonamidophosphates, dithiophosphates, methylphosphonates, 5'-alkylphosphonates, 5'-methylphosphonates, 3'-alkylphosphonates, boron trifluoride, boranophosphates and selenophosphates with 3'-5' linkages or 2'-5' linkages, phosphotriesters, thiocarbonylalkyl phosphotriesters, hydrogen phosphonate linkages, alkylphosphonates, alkylthiophosphonates, arylthiophosphonates, selenophosphates, diselenophosphates, phosphonites, aminophosphates, 3'-alkylaminophosphates, aminoalkylaminophosphates, thiophosphoramidates, piperazine phosphates, anilinothiophosphates, anilinophosphates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazinides, methylenedimethylhydrazinides, methyl acetals, thioacetals, oximes, methyleneiminides, methylenemethyliminides, thioamides, linkages with a nuclear acetyl group, aminoethylglycine, silyl or siloxane linkages, saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl linkages of, for example, 1 to 10 carbons with or without heteroatoms, linkages with a morpholino structure, amides, polyamides (wherein the bases are directly or indirectly linked to the nitrogen heteroatoms of the backbone), and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASO) are antisense oligonucleotides containing phosphorothioate linkages. Methylsulfonamidophosphate antisense oligonucleotides (MsPAASO) are antisense oligonucleotides containing methylsulfonamidophosphate linkages.

[0138] In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate, mesylaminophosphate or thiolphosphonate modification. In some cases, the modified nucleotides include, but are not limited to, 2'-fluoro N3-P5'-aminophosphate.

[0139] In some cases, the modified nucleotides include, but are not limited to, hexitol nucleic acid (or 1',5'-anhydrohexitol nucleic acid (HNA)).

[0140] In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and nucleoside, or modifications of nucleotide analogs at the 3'-end or 5'-end. For example, the 3'-end optionally includes a 3'-cationic group, or the nucleoside at the 3'-end is inverted by using a 3'-3' linkage. In another alternative, the 3'-end is optionally conjugated to an aminoalkyl, such as 3'C5-aminoalkyldT. In an additional alternative, the 3'-end is optionally conjugated to an abasic site, such as a abasic nucleic acid or apyrimidinic nucleic acid site. In some cases, the 5'-end is conjugated to an aminoalkyl, such as a 5'-O-alkylamino substituent. In some cases, the 5'-end is conjugated to an abasic site, such as a abasic nucleic acid or apyrimidinic nucleic acid site.

[0141] In some embodiments, the oligonucleotide molecule comprises one or more of the synthetic nucleotide analogs described herein. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more of the synthetic nucleotide analogs described herein. In some embodiments, the synthetic nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N-methylacetamido (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, 2'-fluoro N3-P5'-aminophosphonate or combinations thereof. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more synthetic nucleotide analogs selected from: 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N-methylacetamido (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, 2'-fluoro N3-P5'-aminophosphonate or combinations thereof. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methyl modified nucleotides. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some cases, the oligonucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiophosphonate nucleotides.

[0142] In some cases, the oligonucleotide molecule comprises at least one of the following: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification. In some cases, the oligonucleotide molecule comprises 100% modification.

[0143] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification.

[0144] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification.

[0145] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification.

[0146] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification.

[0147] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification.

[0148] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification.

[0149] In some cases, the oligonucleotide molecule comprises at least one of the following: about 10% to about 30% modification and about 20% to about 30% modification.

[0150] In some cases, the oligonucleotide molecule comprises about 10% to about 20% modification.

[0151] In some cases, the oligonucleotide molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modification.

[0152] In other cases, the oligonucleotide molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.

[0153] In some embodiments, the oligonucleotide molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 modifications.

[0154] In some cases, the oligonucleotide molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 modified nucleotides.

[0155] In some cases, from about 5% to about 100% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 5% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 10% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 15% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 20% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 25% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 30% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 35% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 40% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 45% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 50% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 55% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 60% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 65% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 70% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 75% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 80% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 85% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 90% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 95% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 96% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 97% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 98% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 99% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein. In some cases, about 100% of the oligonucleotide molecules contain the synthetic nucleotide analogs described herein.In some embodiments, the synthetic nucleotide analogs include LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoro N3-P5'-aminophosphonate, or combinations thereof, modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA).

[0156] In some embodiments, the oligonucleotide molecule comprises from about 1 to about 25 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 1 modification, wherein the modification comprises the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 2 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 3 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 4 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 5 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 6 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 7 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 8 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 9 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 10 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 11 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 12 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 13 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 14 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 15 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 16 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 17 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 18 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 19 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 20 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein.In some embodiments, the oligonucleotide molecule comprises about 21 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 22 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 23 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 24 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 25 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 26 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 27 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 28 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 29 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 30 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 31 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 32 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 33 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 34 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 35 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 36 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 37 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 38 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 39 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein. In some embodiments, the oligonucleotide molecule comprises about 40 modifications, wherein the modifications comprise the synthetic nucleotide analogs described herein.

[0157] In some embodiments, the oligonucleotide molecule is assembled from two separate polynucleotides, where one polynucleotide contains the sense strand of the oligonucleotide molecule and the second polynucleotide contains the antisense strand of the oligonucleotide molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

[0158] In some embodiments, the oligonucleotide molecule contains a sense strand and an antisense strand, where the pyrimidine nucleotides in the sense strand comprise 2'-O-methyl pyrimidine nucleotides, and the purine nucleotides in the sense strand comprise 2'-deoxy purine nucleotides. In some embodiments, the oligonucleotide molecule contains a sense strand and an antisense strand, where the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoro pyrimidine nucleotides, and where the purine nucleotides present in the sense strand comprise 2'-deoxy purine nucleotides.

[0159] In some embodiments, the oligonucleotide molecule contains a sense strand and an antisense strand, where the pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides when present in the antisense strand, and the purine nucleotides are 2'-O-methyl purine nucleotides when present in the antisense strand.

[0160] In some embodiments, the oligonucleotide molecule contains a sense strand and an antisense strand, where the pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides when present in the antisense strand, and where the purine nucleotides when present in the antisense strand comprise 2'-deoxy-purine nucleotides.

[0161] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and at least one of the sense strand and the antisense strand has a plurality (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, etc.) of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides. In some embodiments, at least two, three, four, five, six, or seven of the plurality of 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides. In some embodiments, the consecutive 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, the consecutive 2'-O-methyl or 2'-deoxy-2'-fluoro modified nucleotides are located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the sense strand of the oligonucleotide molecule comprises at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at its 5'-end and / or 3'-end or both. Optionally, in such embodiments, the sense strand of the oligonucleotide molecule comprises at least one, at least two, at least three, at least four 2'-deoxy-2'-fluoro modified nucleotides at the 3'-end of at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at the 5'-end of the polynucleotide, or at the 5'-end of at least four, at least five, at least six consecutive 2'-O-methyl modified nucleotides at the 3'-end of the polynucleotide. Additionally optionally, such at least two, at least three, at least four 2'-deoxy-2'-fluoro modified nucleotides are consecutive nucleotides.

[0162] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and at least one of the sense strand and the antisense strand has a 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand. In some embodiments, at least one of the sense strand and the antisense strand has a 2'-O-methyl modified nucleotide located at the 3'-end of the sense strand and / or the antisense strand. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a purine nucleotide. In some embodiments, the 2'-O-methyl modified nucleotide located at the 5'-end of the sense strand and / or the antisense strand is a pyrimidine nucleotide.

[0163] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and one of the sense strand and the antisense strand has at least two consecutive 2'-deoxy-2'-fluoro-modified nucleotides at the 5' end, while the other strand has at least two consecutive 2'-O-methyl-modified nucleotides at the 5' end. In some embodiments, when the strand has at least two consecutive 2'-deoxy-2'-fluoro-modified nucleotides at the 5' end, the strand further comprises at least two, at least three consecutive 2'-O-methyl-modified nucleotides at the 3' end of the at least two consecutive 2'-deoxy-2'-fluoro-modified nucleotides. In some embodiments, one of the sense strand and the antisense strand has at least two, at least three, at least four, at least five, at least six or at least seven consecutive 2'-O-methyl-modified nucleotides at its 5' end and / or 3' end, and the nucleotides are linked to 2'-deoxy-2'-fluoro-modified nucleotides. In some embodiments, one of the sense strand and the antisense strand has at least four, at least five nucleotides having alternating 2'-O-methyl-modified nucleotides and 2'-deoxy-2'-fluoro-modified nucleotides.

[0164] In some embodiments, an oligonucleotide molecule, such as siRNA, has a formula as shown in Formula III:

[0165] N1N2N3N4N5N6N7N8N9N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 Sense strand (SS)N 21 N 20 N 19 N 18 N 17 N 16 N 15 N 14 N13N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1 Antisense strand (AS),

[0166] wherein each nucleotide represented by N is independently A, U, C or G or a modified nucleobase, such as those provided herein. The N1 nucleotide of the sense strand and the antisense strand represents the 5' end of the corresponding strand. For clarity, although Formula III uses N1, N2, N3, etc. in both the sense strand and the antisense strand, the nucleobases need not be the same and are not intended to be the same. The siRNA shown in Formula III is complementary to the target sequence.

[0167] For example, in some embodiments, the sense strand comprises 2'-O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone at N1 and N2; 2'-fluoro modified nucleotides at N3, N7, N8, N9, N 12 and N 17 ; and 2'-O-methyl modified nucleotides at N4, N5, N6, N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 and N 19 .

[0168] In some embodiments, the antisense strand comprises a vinyl phosphonate moiety linked to N1; 2'-fluoro modified nucleotides with a phosphorothioate (PS) modified backbone at N2; 2'-O-methyl modified nucleotides at N3, N4, N5, N6, N7, N8, N9, N 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 and N 19 ; 2'-fluoro modified nucleotides at N 14 ; and 2'-O-methyl modified nucleotides with a phosphorothioate (PS) modified backbone at N 20 and N 21 .

[0169] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a terminal cap moiety at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the sense strand. In other embodiments, the terminal cap moiety is a reverse deoxy abasic moiety.

[0170] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glycerol modification at the 3'-end of the antisense strand.

[0171] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal cap molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense strand. In other embodiments, one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense strand and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages and / or terminal cap molecules at the 3'-end, 5'-end, or both the 3'-end and 5'-end present in the same or different strands.

[0172] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises from about 1 to about 25, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand; and wherein the antisense strand comprises from about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense strand. In other embodiments, one or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense strand and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without from about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphonate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages and / or terminal capping molecules at the 3'-end, 5'-end, or both the 3'-end and 5'-end present in the same or different strands.

[0173] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, dithiophosphate, phosphonate, aminophosphate or methylsulfonamidophosphate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand and / or antisense strand, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand. In some embodiments, the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate, dithiophosphate, phosphonate, aminophosphate or methylsulfonamidophosphate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more pyrimidine nucleotides of the sense strand and / or antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate, dithiophosphate, phosphonate, aminophosphate or methylsulfonamidophosphate internucleotide linkages and / or a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end present in the same or different strands.

[0174] In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises from about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand; and the antisense strand comprises from about 1 to about 25 or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally a terminal capping molecule at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense strand. In other embodiments, one or more, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense strand and / or the antisense strand are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoro nucleotides, with or without from about 1 to about 5, such as about 1, 2, 3, 4, 5 or more phosphorothioate, phosphorodithioate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages and / or terminal capping molecules at the 3'-end, 5'-end, or both the 3'-end and 5'-end present in the same or different strands.

[0175] In some embodiments, the oligonucleotide molecules described herein are chemically modified short interfering nucleic acid molecules that have from about 1 to about 25, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate, phosphorodithioate, phosphonate, aminophosphonate or methylsulfonamidophosphonate internucleotide linkages in each strand of the oligonucleotide molecule. In some embodiments, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphodiester backbone modification at the 3' end of the antisense strand. Alternatively and / or additionally, the oligonucleotide molecule comprises a sense strand and an antisense strand, and the sense strand comprises a phosphodiester backbone modification at the 5' end of the antisense strand. In some cases, the phosphodiester backbone modification is a phosphorothioate. In some cases, the phosphodiester backbone modification is a phosphorodithioate. In some cases, the phosphodiester backbone modification is a phosphonate. In some cases, the phosphodiester backbone modification is an aminophosphonate. In some cases, the phosphodiester backbone modification is a methylsulfonamidophosphonate. In some embodiments, the sense strand or the antisense strand has three consecutive nucleosides coupled via two phosphorothioate backbones. In some embodiments, the sense strand or the antisense strand has three consecutive nucleosides coupled via two phosphorodithioate backbones. In some embodiments, the sense strand or the antisense strand has three consecutive nucleosides coupled via two phosphonate backbones. In some embodiments, the sense strand or the antisense strand has three consecutive nucleosides coupled via two aminophosphonate backbones. In some embodiments, the sense strand or the antisense strand has three consecutive nucleosides coupled via two methylsulfonamidophosphonate backbones.

[0176] In another embodiment, the oligonucleotide molecules described herein comprise 2'-5' internucleotide linkages. In some cases, the 2'-5' internucleotide linkages are located at the 3' end, 5' end, or both the 3' end and 5' end of one or both of the sequence strands. In other cases, the 2'-5' internucleotide linkages are present at various other positions within one or both of the sequence strands, such as positions of each internucleotide linkage that includes a pyrimidine nucleotide and that has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-5' internucleotide linkages in one or both strands of the oligonucleotide molecule, or positions of each internucleotide linkage that includes a purine nucleotide and that has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-5' internucleotide linkages in one or both strands of the oligonucleotide molecule.

[0177] In some embodiments, the oligonucleotide molecule is a single-stranded molecule that mediates RNAi activity in cells or reconstituted in vitro systems, wherein the oligonucleotide molecule comprises a single-stranded polynucleotide that is complementary to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the oligonucleotide molecule are 2'-deoxy-2'-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and wherein any purine nucleotides present in the oligonucleotide molecule are 2'-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxypurine nucleotides, or multiple purine nucleotides are 2'-deoxypurine nucleotides), and a terminal cap modification, which is optionally present at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the antisense sequence, the oligonucleotide molecule optionally further comprises from about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3'-end of the oligonucleotide molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate or methylsulfonamidate internucleotide linkages, and wherein the oligonucleotide molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0178] In some cases, when compared to natural polynucleic acid molecules and endonucleases, one or more of the synthetic nucleotide analogs described herein are resistant to nucleases such as ribonucleases (e.g., RNase H), deoxyribonucleases (e.g., DNase), or exonucleases (e.g., 5'-3' exonucleases and 3'-5' exonucleases). In some cases, synthetic nucleotide analogs comprising LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoro N3-P5'-aminophosphonate, or combinations thereof modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) are resistant to nucleases such as ribonucleases (e.g., RNase H), deoxyribonucleases (e.g., DNase), or exonucleases (e.g., 5'-3' exonucleases and 3'-5' exonucleases). In some cases, 2'-O-methyl-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-O-aminopropyl-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-deoxy-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-deoxy-2'-fluoro-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-O-aminopropyl (2'-O-AP)-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified oligonucleotide molecules have nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance).In some cases, oligonucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, oligonucleotide molecules modified with 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, oligonucleotide molecules modified with 2'-O-N-methylacetamido (2'-O-NMA) are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA-modified oligonucleotide molecules are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified oligonucleotide molecules are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified oligonucleotide molecules are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholino is nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified oligonucleotide molecules are resistant to nucleases (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, oligonucleotide molecules modified with methylphosphonate are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, oligonucleotide molecules modified with thiophosphonate are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, oligonucleotide molecules containing 2'-fluoro N3-P5'-aminophosphonate are nuclease resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonucleolytic cleavage.

[0179] In some embodiments, one or more of the synthetic nucleotide analogs described herein have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. One or more synthetic nucleotide analogs, comprising LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiophosphonate nucleotide, or 2'-fluoro N3-P5'-aminophosphonate modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA), have increased binding affinity for their mRNA targets. In some cases, 2'-O-methyl modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-aminopropyl modified oligonucleotide molecules have increased binding affinity for mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-deoxy modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-deoxy-2'-fluoro modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules. In some cases, 2'-O-N-methylacetamido (2'-O-NMA) modified oligonucleotide molecules have increased binding affinity for their mRNA targets relative to equivalent native polynucleic acid molecules.In some cases, relative to an equivalent native polynucleic acid molecule, an LNA-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, an ENA-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, a PNA-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, an HNA-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, a morpholino-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, a methylphosphonate nucleotide-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, a thiophosphonate nucleotide-modified oligonucleotide molecule has increased binding affinity for its mRNA target. In some cases, relative to an equivalent native polynucleic acid molecule, an oligonucleotide molecule comprising 2'-fluoro N3-P5'-aminophosphonate has increased binding affinity for an mRNA target. In some cases, the increased affinity is demonstrated by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0180] In some embodiments, the oligonucleotide molecules described herein are chiral pure (or stereopure) polynucleic acid molecules, or polynucleic acid molecules comprising a single enantiomer. In some cases, the oligonucleotide molecule comprises L-nucleotides. In some cases, the oligonucleotide molecule comprises D-nucleotides. In some cases, the oligonucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of its mirror image enantiomer. In some cases, the oligonucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of a racemic mixture.

[0181] In some embodiments, the oligonucleotide molecules described herein are further modified to include an aptamer conjugation moiety. In some cases, the aptamer conjugation moiety is a DNA aptamer conjugation moiety. In some cases, the aptamer conjugation moiety is an Alphamer, which comprises an aptamer moiety that recognizes a specific cell surface target and a moiety that presents a specific epitope for attachment to a circulating antibody.

[0182] In other embodiments, the oligonucleotide molecules described herein are modified to increase their stability. In some embodiments, the oligonucleotide molecules are RNA (e.g., siRNA). In some cases, the oligonucleotide molecules are modified by one or more of the above-described modifications to increase their stability. In some cases, the oligonucleotide molecules are modified at the 2'-hydroxyl position, for example, by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modification or by locking or bridging the ribose conformation (e.g., LNA or ENA). In some cases, the oligonucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the oligonucleotide molecules further include morpholino, PNA, HNA, methylphosphonate nucleotides, thiophosphonate nucleotides, and / or 2'-fluoro N3-P5'-aminophosphates to increase their stability. In some cases, the oligonucleotide molecules are chiral pure (or stereopure) oligonucleotide molecules. In some cases, the chiral pure (or stereopure) oligonucleotide molecules are modified to increase their stability. Suitable modifications to RNA to increase delivery stability will be apparent to those skilled in the art.

[0183] In some embodiments, the oligonucleotide molecule comprises a 2'-modification. In some embodiments, the nucleotides at positions 3, 7, 8, 9, 12, and 17 from the 5'-end of the sense strand of the oligonucleotide molecule are not modified with 2'-O-methyl modification. In some embodiments, the nucleotides at positions 3, 7, 8, 9, 12, and 17 from the 5'-end of the sense strand of the oligonucleotide molecule are modified with 2'-fluoro modification. In some embodiments, the nucleotides at positions 2 and 14 from the 5'-end of the antisense strand of the oligonucleotide molecule are not modified with 2'-O-methyl modification. In some embodiments, the nucleotides at positions 2 and 14 from the 5'-end of the antisense strand of the oligonucleotide molecule are modified with 2'-fluoro modification. In some embodiments, any one of the nucleotides may further comprise a 5'-thiophosphate modification. In some embodiments, the nucleotides at positions 1 and 2 from the 5'-end of the sense strand of the oligonucleotide molecule are modified with 5'-thiophosphate modification. In some embodiments, the nucleotides at positions 1, 2, 20, and 21 from the 5'-end of the antisense strand of the oligonucleotide molecule are modified with 5'-thiophosphate modification. In some embodiments, the 5'-end of the sense or antisense strand of the oligonucleotide molecule may further comprise a vinyl phosphonate modification. In some embodiments, the nucleotide at position 1 from the 5'-end of the antisense strand of the oligonucleotide molecule is modified with vinyl phosphonate modification.

[0184] In some cases, the oligonucleotide molecule is a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some cases, the oligonucleotide molecule is assembled from two separate polynucleotides, wherein one strand is the sense strand and the other strand is the antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand; e.g., wherein the antisense strand and the sense strand form a double helix or double-stranded structure, e.g., wherein the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the oligonucleotide molecule is assembled from a single oligonucleotide, wherein the self-complementary sense and antisense regions of the oligonucleotide molecule are joined by a nucleic acid-based or non-nucleic acid-based linker.

[0185] In some cases, the oligonucleotide molecule is a polynucleotide having a double helix, an asymmetric double helix, a hairpin or an asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleic acid sequence complementary to the nucleic acid sequence of an independent target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleic acid sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the oligonucleotide molecule is a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleic acid sequence complementary to the nucleic acid sequence of the target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleic acid sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed in vivo or in vitro to produce an active oligonucleotide molecule capable of mediating RNAi. In additional cases, the oligonucleotide molecule further comprises a single-stranded polynucleotide comprising a nucleic acid sequence complementary to the nucleic acid sequence of the target nucleic acid molecule or a portion thereof (e.g., wherein such an oligonucleotide molecule does not require the presence of a nucleic acid sequence corresponding to the target nucleic acid sequence or a portion thereof within the oligonucleotide molecule), wherein the single-stranded polynucleotide further comprises a terminal phosphate group, such as a 5'-phosphate or a 5',3'-diphosphate.

[0186] In some cases, the asymmetric hairpin is a linear oligonucleotide molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region, the sense region comprising fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a double helix with a loop. For example, the asymmetric hairpin oligonucleotide molecule comprises an antisense region of sufficient length to mediate RNAi in a cell or in an in vitro system (e.g., about 19 to about 22 nucleotides), and a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, the asymmetric hairpin oligonucleotide molecule further comprises a chemically modified 5' terminal phosphate group. In additional cases, the loop portion of the asymmetric hairpin oligonucleotide molecule comprises nucleotides, non-nucleotides, linker molecules or conjugate molecules.

[0187] In some embodiments, the asymmetric double helix is an oligonucleotide molecule having two independent strands comprising sense and antisense regions, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a double helix. For example, the asymmetric double helix oligonucleotide molecule comprises an antisense region of sufficient length to mediate RNAi in a cell or in an in vitro system (e.g., about 19 to about 22 nucleotides) and a sense region having about 3 to about 19 nucleotides complementary to the antisense region.

[0188] In some cases, a universal base refers to a nucleobase analogue that forms base pairs with each of the natural DNA / RNA bases and has little difference among them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, oxazole carboxamide and nitroazole derivatives known in the art, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole.

[0189] In some embodiments, the dsRNA agent is 5'-phosphorylated or includes a phosphoryl analogue at the 5'-end. The 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate (HO2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate ((HO)2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-monothiophosphate (thiophosphate; (HO)2(S)P--O-5'); 5'-monodithiophosphate (dithiophosphate; (HO)(HS)(S)P--O-5'), 5'-thiophosphate ((HO)2(O)P--S-5'); dithiophosphate [--O2PS2--]; phosphonate [--PO(OH)2--]; aminophosphonate [--O=P(OH)2--]; methanesulfonylaminophosphonate ((CH3)(SO2)(N)P(O)2--O-5'); any additional combination of oxygen / sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphate ((HO)2(O)P—NH-5', (HO)(NH2)(O)P—O-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)--O-5'-), 5'-alkenylphosphonate (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)--O-5'-). In some embodiments, the modification can be placed in the antisense strand of the dsRNA agent.

[0190] Other modifications and modification patterns can be seen, for example, in U.S. Patent No. 10,233,448, which is hereby incorporated by reference. Other modifications and modification patterns can be seen, for example, in Anderson et al., Nucleic Acids Research 2021, 49(16), 9026-9041, which is hereby incorporated by reference. Other modifications and modification patterns can be seen, for example, in PCT Publication No. WO2021 / 030778, which is hereby incorporated by reference. Other modifications and modification patterns can be seen, for example, in PCT Publication No. WO2021 / 030763, which is hereby incorporated by reference.

[0191] In some embodiments, the sequence of the oligonucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.5% complementary to the target sequence of CD40. In some embodiments, the target sequence of CD40 is the nucleic acid sequence of a sequence about 10 - 50 base pairs in length, about 15 - 50 base pairs in length, 15 - 40 base pairs in length, 15 - 30 base pairs in length or 15 - 25 base pairs in length in CD40, wherein the first nucleotide of the target sequence starts at any nucleotide in the coding region or the 5' or 3'-untranslated region (UTR) of the CD40 mRNA transcript. For example, the first nucleotide of the target sequence can be selected such that it starts at a nucleic acid position (nal, numbered from the 5' end of the full-length CD40 mRNA, e.g., the first nucleotide at the 5' end is nal 1) 1, nal 2, nal 3, nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal11, nal 12, nal 13, nal 14, nal 15, nal 15, nal 16, nal 17 or any other nucleic acid position. In some embodiments, the first nucleotide of the target sequence can be selected such that it starts at nal 10 - nal 15, nal 10 - nal20, nal 50 - nal 60, nal 55 - nal 65, nal 75 - nal 85, nal 95 - nal 105, nal 135 - nal 145, nal 155 - nal 165, nal 225 - nal 235, nal 265 - nal 275, nal 275 - nal 245, nal 245 - nal255, nal 285 - nal 335, nal 335 - nal 345, nal 385 - nal 395, nal 515 - nal 525, nal 665 - nal 675, nal 675 - nal 685, nal 695 - nal 705, nal 705 - nal 715, nal 875 - nal 885, nal885 - nal 895, nal 895 - nal 905, nal 1035 - nal 1045, nal 1045 - nal 1055, nal 1125 - nal1135, nal 1135 - nal 1145, nal 1145 - nal 1155, nal 1155 - nal 1165, nal 1125 - nal 1135, nal 1155 - nal 1165, nalPositions within or between 1225-nal 1235, nal 1235-nal 1245, nal 1275-nal 1245, nal1245-nal 1255, nal 1265-nal 1275, nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal 1235, nal 1235-nal 1245, nal 1275-nal 1245, nal 1245-nal 1255, nal 1265-nal1275, nal 1275-nal 1285, nal 1335-nal 1345, nal 1345-nal 1355, nal 1525-nal 1535, nal 1535-nal 1545, nal 1605-nal 1615, nal 1615-c.1625, nal 1625-nal 1635, nal1635-1735, nal 1735-1835, nal 1835-1935, nal.1836-1856, nal 1935-2000, nal 2000-2100, nal 2100-2200, nal 2200-2260, nal 2260-2400, nal 2400-2500, nal 2500-2600, nal 2600-2700, nal 2700-2800, nal 2800-2500, nal 2500-2600, nal 2600-2700, nal2700-2800, nal 2800-2860, etc. In some embodiments, the sequence of CD40 mRNA is provided in the form of an NCBI reference sequence: NM_001250.6 Homo sapiens CD40 molecule (CD40), transcript variant 1, mRNA:

[0192]

[0193]

[0194] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to hybridize therewith and inhibit its expression via RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the cell is a tumor cell, a liver cell, a muscle cell, an immune cell, a heart cell, or a central nervous system cell. In another embodiment, the antisense strand of the dsRNA agent is at least 99%, at least 98%, at least 97%, at least 96%, 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA. In some embodiments, the target RNA is CD40 RNA. In some embodiments, the siRNA molecule is an siRNA that reduces the mRNA expression of CD40. In some embodiments, the siRNA molecule is an siRNA that reduces the mRNA expression of CD40 at a concentration of no more than 200 nM as described herein in the assays described herein and reduces the expression of other RNAs by no more than 50%.

[0195] In some embodiments, the siRNA is linked to a protein, such as an FN3 domain. The siRNA can be linked to multiple FN3 domains that bind to the same or different target proteins. In some embodiments, a linker is linked to the sense strand, which is used to facilitate the linkage of the sense strand to the FN3 domain.

[0196] In some embodiments, provided herein is a compound having the formula (X1) n -(X2) q -(X3) y-A composition of -L-X4, wherein X1 is a first FN3 domain, X2 is a second FN3 domain, X3 is a third FN3 domain or a half-life extending molecule, L is a linker, and X4 is a nucleic acid molecule, such as but not limited to an siRNA molecule, wherein n, q, and y are each independently 0 or 1. In some embodiments, X1, X2, and X3 bind to different target proteins. In some embodiments, y is 0. In some embodiments, n is 1, q is 0, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 1. In some embodiments, X3 increases the overall half-life of the molecule compared to a molecule without X3. In some embodiments, the half-life extending moiety is an FN3 domain that binds to albumin. Examples of such FN3 domains include but are not limited to those described in U.S. Patent Application Publication No. 2017 / 0348397 and U.S. Patent No. 9,156,887, which are hereby incorporated by reference in their entirety. The FN3 domain can be incorporated into other subunits, such as via covalent interactions. In some embodiments, the FN3 domain further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and / or domains, aliphatic chains that bind to serum proteins, transferrin and its fragments and analogs, and Fc regions. The amino acid sequences of human Fc regions are well known and include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE Fc regions. In some embodiments, the FN3 domain can be incorporated into a second FN3 domain that binds to a molecule that extends the half-life of the entire molecule, such as but not limited to any of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin-binding proteins and / or domains and their fragments and analogs.

[0197] In some embodiments, provided herein is a composition having the formula (X1)-(X2)-L-(X4), wherein X1 is a first FN3 domain, X2 is a second FN3 domain, L is a linker, and X4 is a nucleic acid molecule. In some embodiments, X4 is an siRNA molecule. In some embodiments, X1 is an FN3 domain that binds to CD71. In some embodiments, X2 is an FN3 domain that binds to CD71. In some embodiments, X1 and X2 do not bind to the same target protein. In some embodiments, X1 and X2 bind to the same target protein but at different binding sites on the protein. In some embodiments, X1 and X2 bind to the same target protein. In some embodiments, X1 and X2 are FN3 domains that bind to CD71. In some embodiments, the composition does not contain (e.g., is free of) a compound or protein that binds to ASGPR.

[0198] In some embodiments, provided herein are compositions having the formula C-(X1) n -(X2) q [L-X4]-(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0199] In some embodiments, provided herein are compositions having the formula (X1) n -(X2) q [L-X4]-(X3) y -C wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0200] In some embodiments, provided herein are compositions having the formula C-(X1) n -(X2) q [L-X4]L-(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0201] In some embodiments, provided herein are compositions having the formula (X1) n -(X2) q [L-X4]L-(X3) y -C wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is an oligonucleotide molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.

[0202] In some embodiments, provided are compositions or complexes having the formula A1-B1, wherein A1 has the formula C-L1-X s and B1 has the formula X AS -L2-F1, wherein:

[0203] C is a polymer, such as PEG;

[0204] L1 and L2 are each independently a linker;

[0205] X Sis the 5' to 3' oligonucleotide sense strand of a double-stranded siRNA molecule;

[0206] X AS is the 3' to 5' oligonucleotide antisense strand of a double-stranded siRNA molecule;

[0207] F1 is a polypeptide comprising at least one FN3 domain;

[0208] wherein X S and X AS form a double-stranded oligonucleotide molecule to form a composition / complex.

[0209] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula X s and B1 has the formula X AS -L2-F1.

[0210] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula C-L1-X s and B1 has the formula X AS .

[0211] In some embodiments, the sense strand is the sense strand provided herein. In some embodiments, the antisense strand is the antisense strand provided herein. In some embodiments, the sense strand and the antisense strand form a double-stranded siRNA molecule targeting CD40. In some embodiments, the length of the double-stranded oligonucleotide is about 21-23 nucleotide base pairs. In certain embodiments, C is optional.

[0212] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula F1-L1-X s and B1 has the formula X AS -L2-C, wherein:

[0213] F1 is a polypeptide comprising at least one FN3 domain;

[0214] L1 and L2 are each independently a linker;

[0215] C is a polymer, such as PEG;

[0216] X S is the 5' to 3' oligonucleotide sense strand of a double-stranded siRNA molecule;

[0217] X AS is the 3' to 5' oligonucleotide antisense strand of a double-stranded siRNA molecule;

[0218] wherein X S and X ASA double-stranded oligonucleotide molecule is formed to form a composition / complex. In certain embodiments, C is optional.

[0219] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula X s and B1 has the formula X AS -L2-C.

[0220] In some embodiments, a composition or complex having the formula A1-B1 is provided, wherein A1 has the formula F1-L1-X s and B1 has the formula X AS .

[0221] In some embodiments, A1 and B1 interact with each other via hydrogen bonding. In some embodiments, A1 and B1 interact with each other via Watson-Crick base pairing.

[0222] In some embodiments, the composition describes a polymer (polymer moiety C, or just C). In some embodiments, C can be a molecule that extends the half-life of the molecule. In some embodiments, the polymer is a natural or synthetic polymer, consisting of a long chain of branched or unbranched monomers and / or a two-dimensional or three-dimensional cross-linked network of monomers. In some cases, the polymer includes polysaccharides, lignin, rubber, or polyalkylene oxides (such as polyethylene glycol). In some cases, at least one polymer includes, but is not limited to, α-, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polybutylene glycol (PTG), or polyurethanes and mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound and refers to block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some cases, the polymer contains polyalkylene oxide. In some cases, the polymer contains PEG. In some cases, the polymer contains polyethylenimine (PEI) or hydroxyethyl starch (HES).

[0223] In some embodiments, C is a PEG moiety. In some embodiments, the PEG moiety is conjugated to the 5'-end of the oligonucleotide molecule, while the binding moiety is conjugated to the 3'-end of the oligonucleotide molecule. In some embodiments, the PEG moiety is conjugated to the 3'-end of the oligonucleotide molecule, while the binding moiety is conjugated to the 5'-end of the oligonucleotide molecule. In some embodiments, the PEG moiety is conjugated to an internal site of the oligonucleotide molecule. In some embodiments, the PEG moiety, the binding moiety, or a combination thereof is conjugated to an internal site of the oligonucleotide molecule. In some embodiments, the conjugation is a direct conjugation. In some embodiments, the conjugation is via a native ligation.

[0224] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some embodiments, the polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by an average weight (weight average) size and a dispersity. In some embodiments, the monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0225] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0226] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000 or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000 or 100,000 Da. In some embodiments, the molecular weight of C is about 200 Da. In some embodiments, the molecular weight of C is about 300 Da. In some embodiments, the molecular weight of C is about 400 Da. In some embodiments, the molecular weight of C is about 500 Da. In some embodiments, the molecular weight of C is about 600 Da. In some embodiments, the molecular weight of C is about 700 Da. In some embodiments, the molecular weight of C is about 800 Da. In some embodiments, the molecular weight of C is about 900 Da. In some embodiments, the molecular weight of C is about 1000 Da. In some embodiments, the molecular weight of C is about 1100 Da. In some embodiments, the molecular weight of C is about 1200 Da. In some embodiments, the molecular weight of C is about 1300 Da. In some embodiments, the molecular weight of C is about 1400 Da. In some embodiments, the molecular weight of C is about 1450 Da. In some embodiments, the molecular weight of C is about 1500 Da. In some embodiments, the molecular weight of C is about 1600 Da. In some embodiments, the molecular weight of C is about 1700 Da.In some embodiments, the molecular weight of C is about 1800 Da. In some embodiments, the molecular weight of C is about 1900 Da. In some embodiments, the molecular weight of C is about 2000 Da. In some embodiments, the molecular weight of C is about 2100 Da. In some embodiments, the molecular weight of C is about 2200 Da. In some embodiments, the molecular weight of C is about 2300 Da. In some embodiments, the molecular weight of C is about 2400 Da. In some embodiments, the molecular weight of C is about 2500 Da. In some embodiments, the molecular weight of C is about 2600 Da. In some embodiments, the molecular weight of C is about 2700 Da. In some embodiments, the molecular weight of C is about 2800 Da. In some embodiments, the molecular weight of C is about 2900 Da. In some embodiments, the molecular weight of C is about 3000 Da. In some embodiments, the molecular weight of C is about 3250 Da. In some embodiments, the molecular weight of C is about 3350 Da. In some embodiments, the molecular weight of C is about 3500 Da. In some embodiments, the molecular weight of C is about 3750 Da. In some embodiments, the molecular weight of C is about 4000 Da. In some embodiments, the molecular weight of C is about 4250 Da. In some embodiments, the molecular weight of C is about 4500 Da. In some embodiments, the molecular weight of C is about 4600 Da. In some embodiments, the molecular weight of C is about 4750 Da. In some embodiments, the molecular weight of C is about 5000 Da. In some embodiments, the molecular weight of C is about 5500 Da. In some embodiments, the molecular weight of C is about 6000 Da. In some embodiments, the molecular weight of C is about 6500 Da. In some embodiments, the molecular weight of C is about 7000 Da. In some embodiments, the molecular weight of C is about 7500 Da. In some embodiments, the molecular weight of C is about 8000 Da. In some embodiments, the molecular weight of C is about 10,000 Da. In some embodiments, the molecular weight of C is about 12,000 Da. In some embodiments, the molecular weight of C is about 20,000 Da. In some embodiments, the molecular weight of C is about 35,000 Da. In some embodiments, the molecular weight of C is about 40,000 Da. In some embodiments, the molecular weight of C is about 50,000 Da. In some embodiments, the molecular weight of C is about 60,000 Da. In some embodiments, the molecular weight of C is about 100,000 Da.

[0227] In some embodiments, the polyalkylene oxide (e.g., PEG) is discrete PEG, wherein the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide unit. In some embodiments, the discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units. In some embodiments, the dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 2 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 3 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 4 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 5 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 6 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 7 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 8 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 9 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 10 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 11 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 12 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 13 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 14 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 15 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 16 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 17 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 18 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 19 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 20 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 22 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 24 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 26 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 28 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 30 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 35 or more repeating ethylene oxide units.In some embodiments, the dPEG comprises about 40 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 42 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 48 or more repeating ethylene oxide units. In some embodiments, the dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, the dPEG is synthesized in a stepwise manner from pure (e.g., about 95%, 98%, 99% or 99.5%) starting materials into a single molecular weight compound. In some cases, the dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG from QuantaBiodesign, LMD.

[0228] In some embodiments, C is an albumin-binding domain. In some embodiments, the albumin-binding domain specifically binds serum albumin, such as human serum albumin (HSA), to extend the half-life of the domain or another therapeutic agent associated with or linked to the albumin-binding domain. In some embodiments, the human serum albumin-binding domain comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the human serum albumin-binding domain comprises a cysteine (Cys) linked to the C-terminus or N-terminus of the domain. Addition of an N-terminal Met and / or a C-terminal Cys can facilitate expression and / or conjugation to another molecule, which can be another half-life extending molecule, such as PEG, an Fc region, etc.

[0229] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 provided in Table 1. In some embodiments, the albumin binding domain (protein) is isolated. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 901%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, provided that the protein has a substitution corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions when compared to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitutions are located at positions corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.In some embodiments, the provided FN3 domain has at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88 or positions 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91 or 93 of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 or contains a cysteine residue at the C-terminus. Although the positions are listed in series, each position can be selected individually. In some embodiments, cysteine is located at a position corresponding to position 6, 53 or 88. In some embodiments, additional examples of albumin-binding domains can be found in U.S. Patent No. 10,925,932, which is hereby incorporated by reference in its entirety. In some embodiments, additional examples of albumin-binding domains can be found in U.S. Patent Nos. 8,969,289, 9,540,424, 10,221,438, 10,934,572, 10,442,851, 11,203,630, 10,766,946, 11,434,275; and U.S. Publications No. 2022 / 0204589 and 2023 / 0145413; each of which is hereby incorporated by reference in its entirety.

[0230] Table 1: Albumin-Binding Domain Sequences

[0231]

[0232] In some embodiments, C can also be Endoporter, INF-7, TAT, polyarginine, polylysine or amphipathic peptides. These moieties can be used in place of or as a supplement to other half-life extending moieties provided herein. In some embodiments, C can be a molecule that delivers the complex to cells, endosomes or the ER; the molecule is selected from those peptides listed in Table 2.

[0233] Table 2

[0234]

[0235] In some embodiments, L1 can be any linker useful for linking polymer C to sense strand X S or for linking the polypeptide of F1 to sense strand X S In some embodiments, L1 has the following formula:

[0236]

[0237] wherein X S 、X AS and F1 are as defined above.

[0238] In some embodiments, n = 0 - 20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both are absent. In some embodiments, X and Y are independently S. In some embodiments, X and Y are independently present or absent. In some embodiments, the peptide is an enzyme-cleavable peptide, such as but not limited to Val-Cit, Val-Ala, etc.

[0239] In some embodiments, L2 is any linker that can be used to link the polypeptide of F1 to the antisense strand X AS or to link the polymer C to the antisense strand X AS of.

[0240] In some embodiments, L2 has the formula in the following complex:

[0241]

[0242] wherein X AS and F1 are as defined above.

[0243] In some embodiments, n = 0 - 20. In some embodiments, R and R1 are independently methyl. In some embodiments, R and R1 are independently present or both are absent. In some embodiments, X and Y are independently S. In some embodiments, X and Y are independently present or absent. In some embodiments, the peptide is an enzyme-cleavable peptide, such as but not limited to Val-Cit, Val-Ala, etc.

[0244] In some embodiments, the linker is covalently linked to F1 via a cysteine residue present on F1, which can be as follows:

[0245]

[0246] wherein X S is the 5'-to-3' oligonucleotide sense strand of a double-stranded siRNA molecule; X AS is the 3'-to-5' oligonucleotide antisense strand of a double-stranded siRNA molecule; and F1 is a polypeptide comprising at least one FN3 domain, wherein X S and X AS form a double-stranded siRNA molecule.

[0247] In some embodiments, A1 - B1 has the following formula:

[0248]

[0249] Wherein C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, amphipathic peptides or as provided herein; and F1 is a polypeptide comprising at least one FN3 domain. The sense and antisense strands are denoted by the "N" symbol, where each nucleotide denoted by N is independently A, U, C, or G or a modified nucleobase such as those provided herein. The N1 nucleotide of the sense and antisense strands represents the 5' end of the respective strand. For clarity, although Formula III uses N1, N2, N3, etc. in both the sense and antisense strands, the nucleobases need not be the same and are not intended to be the same. The siRNA shown in Formula III is complementary to the target sequence. For example, in some embodiments, the sense strand comprises 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones at N1 and N2; 2'-fluoro modified nucleotides at N3, N7, N8, N9, N 12 and N 17 ; and 2'O-methyl modified nucleotides at N4, N5, N6, N 10 、N 11 、N 13 、N 14 、N 15 、N 16 、N 18 and N 19 .

[0250] In some embodiments, the antisense strand comprises a vinyl phosphonate moiety linked to N1; 2'fluoro modified nucleotides with phosphorothioate (PS) modified backbones at N2; 2'O-methyl modified nucleotides at N3, N4, N5, N6, N7, N8, N9, N 10 、N 11 、N 12 、N 13 、N 15 、N 16 、N 17 、N 18 and N 19 ; 2'fluoro modified nucleotides at N 14 ; and 2'O-methyl modified nucleotides with phosphorothioate (PS) modified backbones at N 20 and N 21 .

[0251] In some embodiments, a compound having the following formula is provided:

[0252]

[0253] In some embodiments, a compound having the following formula is provided:

[0254]

[0255] Wherein F1 is a polypeptide comprising at least one FN3 domain and conjugated to a linker. The linkers shown above are non-limiting examples, and other types of linkers can be used.

[0256] In some embodiments, F1 comprises a polypeptide having the formula (X1) n -(X2) q -(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; wherein n, q, and y are each independently 0 or 1, provided that at least one of n, q, and y is 1. In some embodiments, n, q, and y are each 1. In some embodiments, n and q are 1 and y is 0. In some embodiments, n and y are 1 and q is 0.

[0257] In some embodiments, X1 is an FN3 domain that binds to CD71, such as those provided herein. In some embodiments, X2 is an FN3 domain that binds to CD71. In some embodiments, X1 and X2 are different FN3 domains that bind to CD71. In some embodiments, the binding domains are the same. In some embodiments, X3 is an FN3 domain that binds to human serum albumin. In some embodiments, X3 is an Fc domain with no effector function that extends the half-life of the protein. In some embodiments, X1 is a first FN3 domain that binds to CD71, X2 is a second FN3 domain that binds to CD71, and X3 is an FN3 domain that binds to albumin. Examples of such polypeptides are provided herein and below. In some embodiments, provided herein are compositions having the formula C-(X1) n -(X2) q -(X3) y -L-X4, wherein C is a polymer such as PEG, Endoporter, INF-7, TAT, polyarginine, polylysine, amphipathic peptide, or a peptide provided in Table 2; X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1.

[0258] In some embodiments, provided herein are polypeptides having the formula (X1) n -(X2) q -(X3) y-L-X4-C composition, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, where n, q, and y are each independently 0 or 1.

[0259] In some embodiments, provided herein is a composition having the formula X4-L-(X1) n -(X2) q -(X3) y wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; and X4 is a nucleic acid molecule, where n, q, and y are each independently 0 or 1.

[0260] In some embodiments, provided herein is a composition having the formula C-X4-L-(X1) n -(X2) q -(X3) y wherein C is a polymer; X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; and X4 is a nucleic acid molecule, where n, q, and y are each independently 0 or 1.

[0261] In some embodiments, provided herein is a composition having the formula X4-L-(X1) n -(X2) q -(X3) y -C, wherein X1 is a first FN3 domain; X2 is a second FN3 domain; X3 is a third FN3 domain or a half-life extending molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, where n, q, and y are each independently 0 or 1.

[0262] In some embodiments, the siRNA molecule that binds to CD40 comprises a pair of sequences that can follow the following sequences: sense strand (5'-3') nsnsnnnnNfNfNfnnnnnnnnsnsa or (5'-3') nsnsnnnnNfNfNfnnnnnnnnna; and antisense strand (5'-3') UfsNfsnnnNfnnnnnnnNfnNfnnnsusu, where (n) is 2'-O-Me (methyl), (Nf) is 2'-F (fluorine), and (s) is a phosphorothioate backbone modification. Each nucleotide in the sense strand and the antisense strand can be modified independently or in combination at the ribose and nucleobase positions.

[0263] In some embodiments, the siRNA molecule comprises a pair of sequences from Table 3A, Table 3B, Table 4A or Table 4B. In some embodiments, Tables 5A and 5B depict non-limiting examples of pairs of sequences, wherein the sense strand comprises a linker molecule. In some embodiments, any siRNA molecule provided herein may comprise a linker molecule as disclosed herein.

[0264] In some embodiments, the siRNA molecule comprises a sense strand having a nucleic acid sequence comprising SEQ ID NO: 1890 and an antisense strand having a nucleic acid sequence comprising SEQ ID NO: 2290. In some embodiments, the siRNA molecule comprises a linker at the 3' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the pair of sequences H11 as shown in Table 5B.

[0265] In some embodiments, the siRNA molecule comprises a sense strand having a nucleic acid sequence comprising SEQ ID NO: 1893 and an antisense strand having a nucleic acid sequence comprising SEQ ID NO: 2293. In some embodiments, the siRNA molecule comprises a linker at the 3' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the pair of sequences K11 as shown in Table 5B.

[0266] In some embodiments, the siRNA molecule comprises a sense strand having a nucleic acid sequence comprising SEQ ID NO: 1941 and an antisense strand having a nucleic acid sequence comprising SEQ ID NO: 2051. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the pair of sequences O10 as shown in Table 5B.

[0267] In some embodiments, the siRNA molecule comprises a sense strand having a nucleic acid sequence comprising SEQ ID NO: 1942 and an antisense strand having a nucleic acid sequence comprising SEQ ID NO: 2052. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the pair of sequences P10 as shown in Table 5B.

[0268] In some embodiments, the siRNA molecule comprises a sense strand comprising a nucleic acid sequence of SEQ ID NO: 1944 and an antisense strand comprising a nucleic acid sequence of SEQ ID NO: 2054. In some embodiments, the siRNA molecule comprises a linker at the 5' end of the sense strand. In some embodiments, the linker is C6-NH2-propyl-Mal. In some embodiments, the siRNA molecule comprises the paired sequence R10 as shown in Table 5B.

[0269] Table 3A: siRNA Sense and Antisense Sequences (Modified)

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] Table 3B: siRNA Sense and Antisense Sequences (Unmodified)

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292] Table 4A: Sense and Antisense Sequences of siRNA (Modified)

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] Table 4B: Sense and Antisense Sequences of siRNA (Unmodified)

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] Table 5A: Paired siRNA with Linkers

[0311]

[0312] Table 5B: Paired siRNA with Linkers

[0313]

[0314]

[0315] In some embodiments, the polynucleotides described above include those that do not include 2'-O-methylvinylphosphonate uridine as the 5'-nucleotide on the antisense strand of the siRNA.

[0316] In some embodiments, the polynucleotides are as provided herein. In some embodiments, the polynucleotide comprises a first strand and a second strand that form part of a duplex. In some embodiments, the polynucleotide comprises a sense strand and an antisense strand. In some embodiments, the polynucleotide comprises a sequence shown in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, or Table 5B. In some embodiments, the polynucleotide comprises a sequence shown in Table 3A, Table 4A, or Table 5A without base modifications. In some embodiments, the polynucleotide comprises paired siRNAs as provided herein. In some embodiments, the paired siRNAs are not conjugated to the FN3 domain.

[0317] In some embodiments, the oligonucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, the oligonucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or the physical stability of the duplex formed between the oligonucleotide molecule and the target nucleic acid. Alternatively, the oligonucleotide molecules are produced biotically using an expression vector into which the oligonucleotide molecule has been subcloned in the antisense orientation (i.e., the RNA transcribed from the inserted oligonucleotide molecule will be antisense to the relevant target polynucleic acid molecule).

[0318] In some embodiments, the oligonucleotide molecules are synthesized via a tandem synthesis method in which the two strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which is subsequently cleaved to provide the separate fragments or strands that hybridize and allow purification of the duplex.

[0319] In some cases, the oligonucleotide molecules are also assembled from two different nucleic acid strands or fragments, where one fragment comprises the sense region of the molecule and the second fragment comprises the antisense region of the molecule.

[0320] In some cases, although the internucleotide linkages of the oligonucleotide molecules are chemically modified with phosphorothioates, dithiophosphates, phosphonates, aminophosphonates, or methylsulfonamidophosphonates, the linkages improve stability. Excessive modification sometimes results in reduced toxicity or activity. Thus, when designing nucleic acid molecules, the amount of these internucleotide linkages is minimized in some cases. In such cases, reducing the concentration of these linkages reduces the toxicity of these molecules, increases efficacy, and improves specificity.

[0321] As described herein, in some embodiments, any nucleic acid molecule disclosed herein can be modified to include a linker at the 5' end of the sense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a vinyl phosphonate or a modified vinyl phosphonate at the 5' end of the antisense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a linker at the 3' end of the sense strand of the dsRNA. In some embodiments, any nucleic acid molecule disclosed herein can be modified to include a vinyl phosphonate at the 3' end of the antisense strand of the dsRNA. The linker can be used to link the dsRNA to the FN3 domain. The linker can be covalently linked to, for example, a cysteine residue on the FN3 domain, which can be a naturally occurring cysteine residue or a substituted cysteine residue as described herein and, for example, in U.S. Patent No. 10,196,446, which is hereby incorporated by reference in its entirety.

[0322] In some embodiments, the paired siRNAs of A1-W6 and B7-G11 as shown in Tables 3A and 4A provided above contain a linker at the 3' end of the sense strand. In some embodiments, the paired siRNAs of A1-W6 and B7-G11 as shown in Tables 3A and 4A provided above contain a vinyl phosphonate at the 5' end of the sense strand.

[0323] Non-limiting examples of the structure of linker (L) are shown in Tables 6A and 6B below.

[0324] Table 6A: Exemplary Linker (L) Structures

[0325]

[0326] Table 6B: Exemplary Linker (L) Structures

[0327]

[0328] Other linkers can also be used, such as linkers formed by click chemistry, amide coupling, reductive amination, oxime, and enzymatic coupling (such as transglutaminase and sorting conjugation). The linkers provided herein are exemplary in nature, and other linkers prepared by other such methods can also be used. For example, a linker linked via a phosphate group can be a phosphorothioate or a dithiophosphate.

[0329] When linked to the siRNA, the structure L-(X4) can be represented by one of the following formulas:

[0330]

[0331] Although certain siRNA sequences described herein have certain modified nucleobases, sequences that do not have such modifications are also provided herein. That is, the sequences can include the sequences shown in the tables provided herein without any modifications. In some embodiments, an unmodified siRNA sequence can still include a linker at the 5' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a vinylphosphonate at the 5' end of the antisense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a linker at the 3' end of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecule can be modified to include a vinylphosphonate at the 3' end of the antisense strand of the dsRNA. The linker can be as provided herein.

[0332] In some embodiments, provided is an FN3 protein comprising a polypeptide that binds CD71. In some embodiments, the polypeptide comprises an FN3 domain that binds CD71. In some embodiments, provided is a polypeptide comprising the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849. In some embodiments, the polypeptide that binds CD71 comprises the sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849. The sequence of the CD71 protein to which the polypeptide can bind can be, for example, SEQ ID No: 3 or 4. In some embodiments, the FN3 domain that binds CD71 specifically binds CD71.

[0333] In some embodiments, the FN3 domain that binds CD71 is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon 27 sequence of SEQ ID NO: 2 (LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally having substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponds to SEQ ID NO: 2).

[0334] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849.

[0335] In some embodiments, there is provided a protein comprising a polypeptide having the amino acid sequence of SEQ ID NO: 360. SEQ ID NO: 360 is a consensus sequence based on the sequences of SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, and SEQ ID NO: 364. The sequence of SEQ ID NO: 360 is:

[0336] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 X 13 VKGGX 14 X 15 SX 16 PLX 17 AX 18 FTT,

[0337] wherein X8, X9, X 17 and X 18 are each independently any amino acid other than methionine or proline, and

[0338] X1 is selected from D, F, Y, or H,

[0339] X2 is selected from Y, G, A, or V,

[0340] X3 is selected from I, T, L, A, or H,

[0341] X4 is selected from S, Y, or P,

[0342] X5 is selected from Y, G, Q, or R,

[0343] X6 is selected from G or P,

[0344] X7 is selected from A, Y, P, D, or S,

[0345] X 10 is selected from W, N, S, or E,

[0346] X 11 is selected from L, Y, or G,

[0347] X 12 is selected from D, Q, H, or V,

[0348] X 13 is selected from G or S,

[0349] X 14 is selected from R, G, F, L, or D,

[0350] X 15 is selected from W, S, P, or L, and

[0351] X 16 is selected from T, V, M, or S.

[0352] In some embodiments:

[0353] X1 is selected from D, F, Y, or H,

[0354] X2 is selected from G, A, or V,

[0355] X3 is selected from T, L, A, or H,

[0356] X4 is selected from Y or P,

[0357] X5 is selected from G, Q, or R,

[0358] X6 is selected from G or P,

[0359] X7 is selected from Y, P, D, or S,

[0360] X 10 is selected from W, N, S, or E,

[0361] X 11 is selected from L, Y, or G,

[0362] X 12 is selected from Q, H, or V,

[0363] X 13 is selected from G or S,

[0364] X 14 is selected from G, F, L, or D,

[0365] X 15 is selected from S, P, or L, and

[0366] X 16 is selected from V, M, or S.

[0367] In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 、X 11 、X 12 、X 13 、X 14 、X 15 and X 16 are as shown in the sequence of SEQ ID NO: 361. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 、X 11 、X 12 、X 13 、X 14 、X15 and X 16 as shown in the sequence of SEQ ID NO:362. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 as shown in the sequence of SEQ ID NO:363. In some embodiments, X1, X2, X3, X4, X5, X6, X7, X 10 , X 11 , X 12 , X 13 , X 14 , X 15 and X 16 as shown in the sequence of SEQ ID NO:364.

[0368] In some embodiments, X8, X9, X 17 and X 18 are independently alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, X8, X9, X 17 and X 18 are independently not alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, X8, X9, X 17 and X 18 are independently alanine. In some embodiments, X8, X9, X 17 and X 18 are independently arginine. In some embodiments, X8, X9, X 17 and X 18 are independently asparagine. In some embodiments, X8, X9, X 17 and X 18 are independently aspartic acid. In some embodiments, X8, X9, X 17 and X 18 are independently cysteine. In some embodiments, X8, X9, X 17 and X 18 are independently glutamine. In some embodiments, X8, X9, X 17 and X 18Independently glutamic acid. In some embodiments, X8, X9, X 17 and X 18 are independently glycine. In some embodiments, X8, X9, X 17 and X 18 are independently histidine. In some embodiments, X8, X9, X 17 and X 18 are independently isoleucine. In some embodiments, X8, X9, X 17 and X 18 are independently leucine. In some embodiments, X8, X9, X 17 and X 18 are independently lysine. In some embodiments, X8, X9, X 17 and X 18 are independently phenylalanine. In some embodiments, X8, X9, X 17 and X 18 are independently serine. In some embodiments, X8, X9, X 17 and X 18 are independently threonine. In some embodiments, X8, X9, X 17 and X 18 are independently tryptophan. In some embodiments, X8, X9, X 17 and X 18 are independently tyrosine. In some embodiments, X8, X9, X 17 and X 18 are independently valine.

[0369] In some embodiments, as shown in the sequence of SEQ ID NO:361, except that the positions corresponding to the positions of X8, X9, X 17 and X 18 can be any of the other amino acid residues described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 is not S, and X 18 is not I.

[0370] In some embodiments, as shown in the sequence of SEQ ID NO:362, except that the positions corresponding to the positions of X8, X9, X 17 and X 18 can be any of the other amino acid residues described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 is not S, and X 18 is not I.

[0371] In some embodiments, the sequence is as shown in the sequence of SEQ ID NO: 363, except that the positions corresponding to the positions of X8, X9, X 17 and X 18 may be any of the other amino acid residues described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 is not S, and X 18 is not I.

[0372] In some embodiments, the sequence is as shown in the sequence of SEQ ID NO: 364, except that the positions corresponding to the positions of X8, X9, X 17 and X 18 may be any of the other amino acid residues described above, except that in some embodiments, X8 is not V, X9 is not T, X 17 is not S, and X 18 is not I.

[0373] In some embodiments, the protein comprises a polypeptide having an amino acid sequence that is at least 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360. In some embodiments, the protein is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 360.

[0374] The sequences of SEQ ID NO: 361 - 364 are listed in Table 7 below.

[0375] Table 7: FN3 domain sequences that bind to CD71

[0376]

[0377] The percent identity can be determined by aligning the two sequences using BlastP available via the NCBI website with default parameters.

[0378] As provided herein, in some embodiments, the FN3 domain that binds CD71 binds to human mature CD71 or the extracellular domain of human mature CD71. In some embodiments, human mature CD71 is SEQ ID NO:3, and the extracellular binding domain of human mature CD71 is SEQ ID NO:4, each of which is provided in Table 8 below.

[0379] Table 8: CD71 Sequences

[0380]

[0381]

[0382] As provided herein, the FN3 domain can bind to the CD71 protein. Domains that can also specifically bind to the CD71 protein are also provided, even if not explicitly stated. Thus, for example, the FN3 domain that binds CD71 also encompasses FN3 domain proteins that specifically bind CD71. These molecules can be used, for example, in therapeutic and diagnostic applications and imaging. In some embodiments, polynucleotides encoding the FN3 domains disclosed herein, or their complementary nucleic acids, vectors, host cells, and methods of making and using them are provided. In some embodiments, an isolated FN3 domain that binds or specifically binds CD71 is provided.

[0383] In some embodiments, as determined by surface plasmon resonance or Kinexa methods as practiced by those skilled in the art, the FN3 domain can have a dissociation constant (K -7 ) of less than about 1x10 -8 M, such as less than about 1x10 -9 M, less than about 1x10 -10 M, less than about 1x10 -11 M, less than about 1x10 -12 M, or less than about 1x10 -13 M for binding to CD71. If measured under different conditions (such as osmolality, pH), the affinity of the specific FN3 domain - antigen interaction measured may vary. Thus, the measurement of affinity and other antigen - binding parameters (such as K D , K D , K on , K off ) is performed with standardized solutions of the protein scaffold and antigen and standardized buffers (such as the buffers described herein).

[0384] In some embodiments, in a standard solution ELISA assay, the FN3 domain can bind CD71, with a signal at least 5-fold higher than that obtained with a negative control.

[0385] In some embodiments, the FN3 domain that binds or specifically binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds CD71 comprises a cysteine (Cys) linked to the C-terminus of the FN3 domain. Addition of an N-terminal Met and / or a C-terminal Cys can facilitate expression and / or conjugation to extend the half-life and provide other functions of the molecule.

[0386] The FN3 domain may also contain cysteine substitutions, such as those described in U.S. Patent No. 10,196,446, which is hereby incorporated by reference in its entirety. Briefly, in some embodiments, the polypeptides provided herein may contain at least one cysteine substitution at positions selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domain based on SEQ ID NO:1 or SEQ ID NO:1 of U.S. Patent No. 10,196,446

[0387] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO:2311),

[0388] which is hereby incorporated by reference in its entirety, and equivalent positions in related FN3 domains.

[0389] In some embodiments, the substitution is at residue 6. In some embodiments, the substitution is at residue 8. In some embodiments, the substitution is at residue 10. In some embodiments, the substitution is at residue 11. In some embodiments, the substitution is at residue 14. In some embodiments, the substitution is at residue 15. In some embodiments, the substitution is at residue 16. In some embodiments, the substitution is at residue 20. In some embodiments, the substitution is at residue 30. In some embodiments, the substitution is at residue 34. In some embodiments, the substitution is at residue 38. In some embodiments, the substitution is at residue 40. In some embodiments, the substitution is at residue 41. In some embodiments, the substitution is at residue 45. In some embodiments, the substitution is at residue 47. In some embodiments, the substitution is at residue 48. In some embodiments, the substitution is at residue 53. In some embodiments, the substitution is at residue 54. In some embodiments, the substitution is at residue 59. In some embodiments, the substitution is at residue 60. In some embodiments, the substitution is at residue 62. In some embodiments, the substitution is at residue 64. In some embodiments, the substitution is at residue 70. In some embodiments, the substitution is at residue 88. In some embodiments, the substitution is at residue 89. In some embodiments, the substitution is at residue 90. In some embodiments, the substitution is at residue 91. In some embodiments, the substitution is at residue 93.

[0390] A cysteine substitution at a position in a domain or protein involves replacing an existing amino acid residue with a cysteine residue. In some embodiments, instead of a substitution, a cysteine is inserted into a sequence adjacent to the positions listed above. Other examples of cysteine modification can be found, for example, in U.S. Patent Application Publication No. 2017 / 0362301, which is hereby incorporated by reference in its entirety. Alignment of sequences can be performed, for example, using BlastP with default parameters on the NCBI website.

[0391] In some embodiments, a cysteine residue is inserted at any position in a domain or protein.

[0392] In some embodiments, the FN3 domain that binds CD71 is internalized into cells. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a detectable label or therapeutic agent into cells. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent into cells. The cytotoxic agent can act as a therapeutic agent. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of any detectable label, therapeutic agent, and / or cytotoxic agent disclosed herein into cells. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of oligonucleotides into cells. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a central nervous system cell. In some embodiments, the cell is a heart cell. In some embodiments, the therapeutic agent is an siRNA molecule as provided herein. The FN3 domain that binds CD71 conjugated to a detectable label can be used to evaluate the expression of CD71 in vivo or in vitro on, for example, samples of tumor tissue. The FN3 domain that binds CD71 conjugated to a detectable label can be used to evaluate the expression of CD71 in vivo or in vitro on samples of blood, immune cells, or muscle cells.

[0393] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 360-644, 663-672, or 1395-1849.

[0394] In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:365. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:366. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:367. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:368. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:369. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:370. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:371. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:372. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:373. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:374. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:375. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:376. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:377. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:378. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:379. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:380. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:381. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:382. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:383. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:384.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:385. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:386. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:387. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:388. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:389. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:390. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:391. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:392. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:393. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:394. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:395. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:396. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:397. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:398. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:399. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:400. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:401. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:402. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:403. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:404.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 405. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 407. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 409. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 410. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 414. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 418. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 422. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 423. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 424.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 425. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 427. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 429. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 431. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 432. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 433. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 435. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 437. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 439. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 441. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 443. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 444.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 445. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 446. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 447. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 448. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 449. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 450. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 451. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 453. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 455. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 457. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 459. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 461. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 464.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 465. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 471. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 479. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 484.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 487. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 489. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 491. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 501. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 504.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:505. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:506. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:507. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:508. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:509. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:510. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:511. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:512. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:513. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:514. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:515. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:516. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:517. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:518. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:519. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:520. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:521. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:522. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:523. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:524.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:525. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:526. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:527. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:528. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:529. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:530. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:531. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:532. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:533. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:534. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:535. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:536. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:537. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:538. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:539. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:540. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:541. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:542. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:543. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:544.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:545. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:546. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:547. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:548. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:549. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:550. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:551. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:552. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:553. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:554. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:555. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:556. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:557. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:558. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:559. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:560. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:561. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:562. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:563. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:564.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:565. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:566. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:567. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:568. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:569. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:570. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:571. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:572. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:573. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:574. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:575. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:576. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:577. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:578. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:579. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:580. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:581. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:582. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:583. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:584.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:585. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:586. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:587. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:588. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:589. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:590. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:591. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:592. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:593. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:594. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:595. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:596. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:597. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:598. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:599. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:600. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:601. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:602. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:603. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:604.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 605. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 606. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 612. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 613. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 615. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 616. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 619. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 620. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 621. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 622. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 623. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 624.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 625. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 626. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 627. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 629. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 630. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 631. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 633. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 634. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 635. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 636. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 637. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 638. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 639. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 640. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 641. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 642. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 643. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 644.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 663. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 664. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 665. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 667. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 668. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 669. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 670. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 671. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 672. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1395. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1396. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1397. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1398. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1399. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1400. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1401. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1402. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1403. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1404.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1405. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1406. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1407. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1408. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1409. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1410. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1411. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1412. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1413. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1414. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1415. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1416. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1417. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1418. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1419. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1420. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1421. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1422. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1423. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1424.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1425. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1426. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1427. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1428. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1429. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1430. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1431. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1432. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1433. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1434. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1435. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1436. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1437. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1438. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1439. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1440. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1441. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1442. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1443. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1444.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1445. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1446. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1447. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1448. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1449. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1450. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1451. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1452. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1453. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1454. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1455. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1456. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1457. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1458. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1459. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1460. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1461. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1462. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1463. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1464.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1465. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1466. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1467. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1468. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1469. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1470. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1471. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1472. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1473. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1474. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1475. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1476. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1477. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1478. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1479. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1480. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1481. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1482. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1483. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1484.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1485. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1486. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1487. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1488. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1489. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1490. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1491. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1492. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1493. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1494. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1495. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1496. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1497. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1498. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1499. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1500. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1501. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1502. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1503. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1504.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1505. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1506. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1507. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1508. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1509. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1510. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1511. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1512. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1513. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1514. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1515. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1516. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1517. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1518. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1519. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1520. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1521. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1522. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1523. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1524.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1525. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1526. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1527. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1528. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1529. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1530. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1531. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1532. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1533. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1534. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1535. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1536. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1537. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1538. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1539. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1540. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1541. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1542. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1543. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1544.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1545. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1546. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1547. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1548. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1549. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1550. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1551. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1552. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1553. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1554. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1555. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1556. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1557. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1558. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1559. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1560. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1561. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1562. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1563. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1564.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1565. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1566. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1567. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1568. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1569. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1570. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1571. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1572. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1573. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1574. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1575. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1576. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1577. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1578. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1579. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1580. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1581. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1582. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1583. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO:1584.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1585. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1586. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1587. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1588. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1589. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1590. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1591. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1592. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1593. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1594. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1595. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1596. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1597. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1598. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1599. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1600. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1601. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1602. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1603. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1604.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1605. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1606. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1607. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1608. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1609. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1610. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1611. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1612. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1613. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1614. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1615. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1616. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1617. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1618. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1619. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1620. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1621. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1622. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1623. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1624.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1625. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1626. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1627. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1628. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1629. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1630. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1631. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1632. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1633. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1634. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1635. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1636. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1637. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1638. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1639. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1640. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1641. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1642. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1643. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1644.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1645. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1646. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1647. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1648. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1649. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1650. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1651. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1652. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1653. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1654. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1655. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1656. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1657. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1658. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1659. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1660. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1661. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1662. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1663. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1664.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1665. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1666. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1667. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1668. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1669. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1670. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1671. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1672. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1673. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1674. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1675. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1676. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1677. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1678. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1679. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1680. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1681. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1682. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1683. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:1684.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1685. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1686. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1687. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1688. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1689. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1690. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1691. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1692. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1693. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1694. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1695. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1696. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1697. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1698. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1699. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1700. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1701. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1702. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1703. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1704.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1705. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1706. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1707. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1708. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1709. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1710. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1711. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1712. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1713. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1714. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1715. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1716. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1717. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1718. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1719. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1720. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1721. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1722. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1723. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1724.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1725. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1726. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1727. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1728. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1729. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1730. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1731. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1732. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1733. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1734. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1735. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1736. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1737. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1738. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1739. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1740. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1741. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1742. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1743. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1744.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1745. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1746. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1747. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1748. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1749. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1750. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1751. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1752. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1753. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1755. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1756. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1757. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1758. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1759. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1760. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1761. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1762. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1763. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1764.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1765. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1766. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1767. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1768. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1769. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1770. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1771. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1772. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1773. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1774. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1775. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1776. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1777. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1778. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1779. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1780. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1781. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1782. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1783. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1784.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1785. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1786. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1787. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1788. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1789. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1790. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1791. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1792. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1793. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1794. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1795. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1796. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1797. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1798. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1799. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1800. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1801. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1802. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1803. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1804.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1805. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1806. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1807. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1808. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1809. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1810. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1811. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1812. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1813. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1814. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1815. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1816. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1817. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1818. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1819. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1820. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1821. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1822. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1823. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1824.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1825. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1826. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1827. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1828. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1829. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1830. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1831. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1832. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1833. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1834. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1835. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1836. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1837. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1838. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1839. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1840. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1841. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1842. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1843. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 1844.In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1845. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1846. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1847. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1848. In some embodiments, the isolated CD71-binding FN3 domain comprises the amino acid sequence of SEQ ID NO: 1849.

[0395] In some embodiments, the isolated CD71-binding FN3 domain comprises an initiating methionine (Met) linked to the N-terminus of the molecule.

[0396] In some embodiments, the isolated CD71-binding FN3 domain comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of the amino acid sequences of SEQ ID NOs: 365-644, 663-672, or 1395-1849. The percent identity can be determined by aligning the two sequences using BlastP available via the NCBI website with default parameters. The sequences of the CD71-binding FN3 domains are shown, for example, in Table 9. These sequences are shown with an N-terminal methionine. The sequences of the domains can also be utilized without the N-terminal methionine. A table of such sequences is not provided merely to avoid duplicating sequences that are nearly identical, but those skilled in the art can immediately envision the sequences without the N-terminal methionine provided herein, and the present disclosure should be understood and interpreted to include such sequences.

[0397] Table 9: CD71-binding FN3 domain sequences

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446] Without being bound by any particular theory, in some embodiments, the FN3 domain attached to a nucleic acid molecule can be used to target the delivery of a therapeutic agent to cells expressing a binding partner for one or more FN3 domains and to direct the intracellular accumulation of the nucleic acid molecule therein. This can enable the siRNA molecule to interact appropriately with the cellular machinery to inhibit the expression of the target gene, enhance efficacy, and in some embodiments, also avoid the toxicity that may result from the non-targeted administration of the same siRNA molecule.

[0447] The FN3 domains described herein that bind to a specific target protein can be produced as monomers, dimers, or multimers, for example as a means of increasing valency and thus the affinity of target molecule binding, or to generate bispecific or multispecific scaffolds that bind two or more different target molecules simultaneously. Dimers and multimers can be produced by linking monospecific, bispecific, or multispecific protein scaffolds, for example by including amino acid linkers such as those containing polyglycine, glycine and serine, or alanine and proline.

[0448] Thus, as provided herein, different FN3 domains linked to siRNA molecules can also be conjugated or linked to another FN3 domain that binds a different target. The linker can be a flexible linker. The linker can be a short peptide sequence such as those described herein. For example, the linker can be a G / S or G / A linker, etc. As provided herein, the linker can be, for example, the linker shown in Table 10.

[0449] Table 10: Exemplary peptide linker sequences

[0450] SEQ ID NO Sequence 645 <![CDATA[(GS)2]]> 646 <![CDATA[(GGGS)2]]> 647 <![CDATA[(GGGGS) 1-5 > 648 <![CDATA[(GGGGS)5]]> 649 <![CDATA[(GGGGA) 1-5 > 650 <![CDATA[(AP) 1-20 > 651 <![CDATA[(AP) 2-20 > 652 <![CDATA[(AP)2]]> 653 <![CDATA[(AP)5]]> 654 <![CDATA[(AP) 10 > 655 <![CDATA[(AP) 20 > 656 <![CDATA[A(EAAAK)5AAA]]> 657 <![CDATA[(EAAAK) 1-5 > 658 EAAAKEAAAKEAAAKEAAAK 659 GGGGSGGGGSGGGGSGGGGS 660 APAPAPAPAP 661 EAAAK

[0451] In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 645. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 646. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 647. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 648. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 649. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 650. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 651. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 652. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 653. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 654. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 655. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 656. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 657. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 658. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 659. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 660. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of SEQ ID NO: 661. In some embodiments, the FN3 domain comprising two FN3 domains linked by a linker has the amino acid sequence of any one of SEQ ID No: 645 - 661.

[0452] Dimers and multimers can be linked to each other in the N-to-C direction. The linking of polypeptides into novel linked fusion polypeptides using both naturally occurring and synthetic peptide linkers is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson and Sauer, Biochemistry 35, 109-116, 1996; U.S. Patent No. 5,856,456). The linkers described in this paragraph can also be used to link the domains provided in the formulas herein and above.

[0453] Half-life extension moiety

[0454] In some embodiments, the FN3 domain can also be incorporated into other subunits, for example, via covalent interactions. In some embodiments, the FN3 domain also contains a half-life extension moiety. Exemplary half-life extension moieties are albumin, albumin variants, albumin-binding proteins and / or domains, one or more aliphatic chains that bind to serum proteins, transferrin and its fragments and analogs, and Fc regions. The amino acid sequences of human Fc regions are well known and include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE Fc regions. In some embodiments, the FN3 domain binds to albumin, albumin variants, albumin-binding proteins and / or domains and their fragments and analogs, thereby extending the half-life of the entire molecule.

[0455] In some embodiments, the albumin binding domain comprises the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain (protein) is isolated. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the albumin binding domain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 901%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, provided that the protein has a substitution corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid. In some embodiments, the isolated albumin binding domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions when compared to the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the substitutions are located at positions corresponding to position 10 of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.In some embodiments, the provided FN3 domain has at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88 of SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 or at residue positions 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89 or 90 or contains a cysteine residue at the C-terminus. Although the positions are listed in series, each position can also be selected individually. In some embodiments, cysteine is located at a position corresponding to position 6, 53 or 88. In some embodiments, additional examples of albumin-binding domains can be found in U.S. Patent No. 10,925,932, which is hereby incorporated by reference.

[0456] All or a portion of the antibody constant region can be linked to the FN3 domain to confer antibody-like properties, particularly those associated with the Fc region, such as Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), and can be further modified by modifying residues in the Fc responsible for these activities (for a review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0457] Additional moieties can be incorporated into the FN3 domain, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000; fatty acids and fatty acid esters of different chain lengths, such as laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, suberic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc.; polylysine; octane; carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) to obtain desired properties. These moieties can be directly fused to the protein backbone coding sequence and can be produced by standard cloning and expression techniques. Alternatively, the moieties can be linked to the recombinantly produced molecules disclosed herein using well-known chemical coupling methods.

[0458] PEG moieties can be added to the FN3 domain, which is, for example, by incorporating a cysteine residue at the C-terminus of the molecule, or engineering cysteine to a residue position away from the binding surface of the molecule, and using well-known methods to link the PEG group to the cysteine.

[0459] The function of FN3 domains incorporating additional moieties can be compared by several well-known assays. For example, properties altered due to the incorporation of an Fc domain and / or Fc domain variant can be determined using a soluble form of a receptor (e.g., an FcγRI, FcγRII, FcγRIII, or FcRn receptor) in an Fc receptor binding assay, or a cell-based assay measuring well-known measures such as ADCC or CDC, or by evaluating the pharmacokinetic properties of the molecules disclosed herein in an in vivo model.

[0460] The compositions provided herein can be prepared by making FN3 proteins and nucleic acid molecules and linking them together. Techniques for linking proteins to nucleic acid molecules are known and any method can be used. For example, in some embodiments, the nucleic acid molecule is modified with a linker (e.g., a linker provided herein) and then the protein is mixed with the nucleic acid molecule comprising the linker to form the composition. For example, in some embodiments, an FN3 domain is conjugated to siRNA via a cysteine using thiol-maleimide chemistry. In some embodiments, an FN3 domain containing a cysteine can be reduced with a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) in, for example, phosphate buffered saline (or any other suitable buffer) to produce a free thiol. Then, in some embodiments, the FN3 domain containing the free thiol is mixed with a maleimide-linked modified siRNA duplex and incubated under conditions to form a linkage complex. In some embodiments, the mixture is incubated at room temperature (RT) for 0 - 5 hours or about 1, 2, 3, 4, or 5 hours. The reaction can be quenched, for example, with N-ethylmaleimide. In some embodiments, the conjugate can be purified using affinity chromatography and ion exchange. Other methods can also be used and this is merely a non-limiting embodiment.

[0461] Methods for making FN3 proteins are known and any method can be used to produce the protein. Examples are provided in the references incorporated herein by reference.

[0462] In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NO: 365 - 644 or 663 - 672, wherein a histidine tag has been attached to the N-terminus or C-terminus of the polypeptide to facilitate purification. In some embodiments, the histidine tag (His-tag) comprises six histidine residues (SEQ ID NO: 662). In other embodiments, the His-tag is linked to the FN3 domain by at least one glycine residue or about 2 to about 4 glycine residues. Thus, after purifying the FN3 domain and cleaving the His-tag from the polypeptide, one or more glycines may remain at the N-terminus or C-terminus. In some embodiments, if the His-tag is removed from the N-terminus, all glycines are removed. In some embodiments, if the His-tag is removed from the C-terminus, one or more glycines are retained.

[0463] In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NO: 365 - 644 or 663 - 672, wherein the N-terminal methionine is retained after purification of the FN3 domain. In some embodiments, the FN3 domain that specifically binds to CD71 comprises the amino acid sequence of SEQ ID NO: 365 - 644 or 663 - 672, wherein the N-terminal methionine is not retained after purification of the FN3 domain.

[0464] For example, as described herein, in some embodiments, the amino acid sequence of methionine-free SEQ ID NO: 570 is as follows:

[0465] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYAEPRPDGEAILLQVPGSCRSYDLTGLKPGTEYSVLIHGVKGGLLSSPLTAIFTT (SEQ ID NO: 2310)

[0466] As provided herein, the FN3 domain can be linked to an siRNA molecule. Although certain FN3 domains are shown with a methionine, it should be understood that the FN3 domain can be linked to the siRNA in the absence of an N-terminal methionine. Further, those skilled in the art will appreciate that in the absence of an N-terminal methionine, the numbering of the cysteine residue positions provided herein will shift to the next lower residue.

[0467] For example, the amino acid sequence of the FN3 domain can be, for example, the amino acid sequences provided herein, including but not limited to amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or identical to the amino acid sequences of SEQ ID NO:570 or SEQ ID NO:2310, and can be linked to paired siRNAs as provided herein. In some embodiments, the paired siRNAs comprise a sense strand and an antisense strand. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2110 or a modified form thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2220 or a modified form thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2113 or a modified form thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2223 or a modified form thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:2111 or a modified form thereof, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2221 or a modified form thereof. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1890 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2290. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1893 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2293. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1941 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2051. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1942 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2052. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1944 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:2054. In some embodiments, the sense strand and antisense strand pair (paired siRNA) are as provided herein.

[0468] Kit

[0469] In some embodiments, a kit is provided that comprises the compositions described herein. The kit can be used for therapeutic purposes and as a diagnostic kit. In some embodiments, the kit comprises an FN3 domain conjugated to a nucleic acid molecule.

[0470] Use of the conjugate

[0471] The compositions provided herein can be used for diagnosing, monitoring, modulating, treating, alleviating human diseases or specific lesions of cells, tissues, organs, body fluids or the host in general, helping to prevent their onset or relieve their symptoms.

[0472] In some embodiments, the FN3 domain can facilitate delivery to activated lymphocytes, dendritic cells or other immune cells for treating immune diseases. Thus, in some embodiments, the FN3 domain that binds CD71 is targeted to immune cells. In some embodiments, the FN3 domain that binds CD71 is targeted to B cells. In some embodiments, the FN3 domain that binds CD71 is targeted to T cells. In some embodiments, the FN3 domain that binds CD71 is targeted to dendritic cells. In some embodiments, the FN3 domain that binds CD71 is targeted to monocytes. In some embodiments, the FN3 domain that binds CD71 does not have an anti-proliferative effect on immune cells. For example, in some embodiments, the FN3 domain that binds CD71 does not have an anti-proliferative effect on B cells, T cells, dendritic cells, monocytes or any combination thereof.

[0473] In some embodiments, methods of treating an autoimmune disease in a subject in need thereof are provided. In some embodiments, the methods comprise administering to the subject a polypeptide or pharmaceutical composition that binds CD71. In some embodiments, the polypeptide is an FN3 domain that binds CD71. In some embodiments, the polypeptide comprises an amino acid sequence such as SEQ ID NO: 361 - 644 or 663 - 672, or a polypeptide as provided herein linked or conjugated to a therapeutic agent. In some embodiments, a method of treating an autoimmune disease in a subject, the method comprising administering to the subject an FN3 domain that binds CD71, and the FN3 domain is conjugated to a therapeutic agent (such as a cytotoxic agent; an oligonucleotide, such as siRNA, ASO, etc.; an FN3 domain that binds another target; etc.).

[0474] In some embodiments, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Sjogren's syndrome, myositis, lupus nephritis, neuroinflammatory diseases (such as multiple sclerosis) or preventing solid organ transplant rejection.

[0475] In some embodiments, methods are provided for reducing the expression of a target gene in a cell. In some embodiments, the method comprises delivering a composition or pharmaceutical composition as provided herein to the cell. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the target gene is CD40. However, the target gene can be any target gene, as the evidence provided herein shows that siRNA molecules can be effectively delivered when conjugated to an FN3 domain. In some embodiments, the siRNA targeting CD40 is linked to an FN3 domain. In some embodiments, the FN3 polypeptide (domain) binds CD71. In some embodiments, the FN3 polypeptide is as provided herein or as provided in PCT Application No. PCT / US20 / 55509, U.S. Application No. 17 / 070,337, PCT Application No. PCT / US20 / 55470, or U.S. Application No. 17 / 070,020, each of which is hereby incorporated by reference in its entirety. In some embodiments, the siRNA is not conjugated to an FN3 domain. In some embodiments, the method of reducing the expression of the target gene results in a reduction in the expression of the target gene of about 99%, 90 - 99%, 50 - 90%, or 10 - 50%.

[0476] In some embodiments, a method of reducing CD40 expression is provided. In some embodiments, the reduced expression is the expression (amount) of CD40 mRNA. In some embodiments, the method of reducing CD40 expression results in a reduction in CD40 expression of about 99%, 90 - 99%, 50 - 90%, or 10 - 50%. In some embodiments, the reduced expression is the expression (amount) of CD40 protein. In some embodiments, the protein being reduced is CD40 protein. In some embodiments, the reduction of CD40 protein occurs in immune cells. In some embodiments, the reduction of CD40 protein occurs in B cells. In some embodiments, the reduction of CD40 protein occurs in T cells. In some embodiments, the reduction of CD40 protein occurs in dendritic cells. In some embodiments, the method comprises delivering an siRNA molecule targeting CD40 as provided herein to a cell. In some embodiments, the siRNA is conjugated to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer of two FN3 domains that bind CD71. In some embodiments, the FN3 domains are identical. In some embodiments, the two FN3 domains are different, i.e., bind different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the method comprises administering to a subject (patient) an siRNA molecule targeting CD40, such as those provided herein. In some embodiments, the siRNA molecule targeting CD40 administered to the subject is conjugated or linked to an FN3 domain. In some embodiments, the FN3 domain is an FN3 domain that binds CD71. In some embodiments, the FN3 domain is as provided herein. In some embodiments, the FN3 domain is a dimer of two FN3 domains that bind CD71. In some embodiments, the FN3 domains are identical. In some embodiments, the two FN3 domains are different, i.e., bind different regions or amino acid residues of CD71, i.e., different epitopes. In some embodiments, the CD71 binding domain is a polypeptide as provided herein.

[0477] In some embodiments, methods are provided for delivering siRNA molecules to the cells of a subject. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition comprising a composition as provided herein. In some embodiments, the cells are CD71-positive cells. The term "positive cell" with respect to a protein refers to a cell that expresses the protein. In some embodiments, the protein is expressed on the cell surface. In some embodiments, the cells are tumor cells, liver cells, immune cells, heart cells, muscle cells, CNS cells, or cells within the blood-brain barrier. In some embodiments, the cells are immune cells. In some embodiments, the cells are B cells. In some embodiments, the cells are T cells. In some embodiments, the cells are dendritic cells. In some embodiments, the siRNA molecule downregulates the expression of a target gene in the cell. In some embodiments, the target gene is CD40.

[0478] In some embodiments, methods are provided for reducing one or more serum cytokines in a subject. In some embodiments, the methods comprise administering an siRNA molecule. In some embodiments, the siRNA molecule downregulates the expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the one or more cells are CD71-positive cells. In some embodiments, the one or more cells are immune cells. In some embodiments, the cells are B cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are T cells. In some embodiments, the one or more serum cytokines comprise IFN-γ, IL-6, TNF-α, IL-12, IP-10, and / or RANTES or any combination thereof. In some embodiments, the one or more serum cytokines comprise IFN-γ. In some embodiments, the one or more serum cytokines comprise IL-6. In some embodiments, the one or more serum cytokines comprise TNF-α. In some embodiments, the one or more serum cytokines comprise IL-12. In some embodiments, the one or more serum cytokines comprise IP-10. In some embodiments, the one or more serum cytokines comprise RANTES.

[0479] In some embodiments, methods are provided for reducing or inhibiting cell migration. In some embodiments, the methods comprise contacting a cell with an siRNA molecule. In some embodiments, the siRNA molecule downregulates the expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the cell is a CD71-positive cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the methods comprise reducing or inhibiting the migration of cells from the blood to a tissue. In some embodiments, the methods comprise reducing or inhibiting the migration of cells from the blood to lymphoid organ tissue. In some embodiments, the methods comprise selectively reducing or inhibiting the migration of B cells and / or dendritic cells without reducing or inhibiting the migration of T cells.

[0480] In some embodiments, methods are provided for inhibiting margination. In some embodiments, the methods comprise contacting a cell with an siRNA molecule. In some embodiments, the siRNA molecule downregulates the expression of a target gene in the cell. In some embodiments, the target gene is CD40. In some embodiments, the cell is a CD71-positive cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the methods comprise reducing or inhibiting the margination of cells from the interior of a blood vessel to the blood vessel wall. In some embodiments, the methods comprise selectively reducing or inhibiting the margination of B cells and / or dendritic cells without reducing or inhibiting the margination of T cells.

[0481] In some embodiments, the compositions or pharmaceutical compositions provided herein may be administered alone or in combination with other therapeutic agents, i.e., simultaneously or sequentially.

[0482] "Treat" or "treatment" refers to therapeutic treatment and prophylactic measures, wherein the goal is to prevent or slow down (alleviate) an undesired physiological change or condition, such as the development or spread of cancer. In some embodiments, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete remission), whether detectable or not. "Treatment" may also mean prolonging survival as compared to the expected survival of those not receiving treatment. Those in need of treatment include those who already have a disorder or condition, as well as those who are predisposed to a disorder or condition or in whom a disorder or condition is to be prevented.

[0483] "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result in a required dose and period of time. The therapeutically effective amount of the compositions provided herein can vary depending on factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective amount are improvement in the patient's health condition, reduction or shrinkage of tumor size, arrest or slowing of tumor growth, and / or absence of metastasis of cancer cells to other parts of the body.

[0484] Administration / Pharmaceutical Composition

[0485] In some embodiments, a pharmaceutical composition comprising the compositions provided herein and a pharmaceutically acceptable carrier is provided. For therapeutic use, the compositions can be formulated as a pharmaceutical composition containing an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% normal saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques, such as filtration. The compositions can optionally contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants, and coloring agents, etc. The concentration of the molecules disclosed herein in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight, usually at least about 1% to up to 15% or 20%, and will be selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration chosen. Suitable vehicles and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B., ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691 - 1092 (see especially pp. 958 - 9...

Claims

1. A composition, the composition comprising an siRNA molecule targeting the CD40 gene, the molecule comprising a sense strand and an antisense strand, wherein: the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1890, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2290; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1893, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2293; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1941, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2051; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1942, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2052; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1944, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2054; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 2110 or a modified form thereof, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2220 or a modified form thereof; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 2113 or a modified form thereof, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2223 or a modified form thereof; the sense strand comprises the nucleic acid sequence of SEQ ID NO: 2111 or a modified form thereof, and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2221 or a modified form thereof; or a sense strand and antisense strand pair (paired siRNA) as provided herein.

2. The composition according to claim 1, wherein the siRNA molecule further comprises a linker covalently linked to the sense strand or the antisense strand.

3. The composition according to claim 2, wherein the linker is linked to the 5' end or the 3' end of the sense strand or the antisense strand.

4. The composition according to claim 1, wherein the siRNA molecule further comprises a vinyl phosphonate modification on the sense strand or the antisense strand.

5. The composition according to claim 4, wherein the vinyl phosphonate modification is linked to the 5' end or the 3' end of the sense strand or the antisense strand.

6. The composition according to claim 1, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NO: 1890, 1893, 1941, 1942, 1944, 46-178, 312-331, 1850, 1851, 1891, 1892, 1894-1928, 1932-1940, 1943, 1945-1959, 2298, 2302, 2304, 352-356, 673-805, 939-958, 2070, 2071, 2110-2148, 2152-2179, 2300, 2306 and 2308 or a modified form thereof.

7. The composition according to claim 1, wherein the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NO: 2290, 2293, 2051, 2052, 2054, 179 - 311, 332 - 351, 1960, 1961, 2000 - 2038, 2042 - 2050, 2053, 2055 - 2069, 2291, 2292, 2294 - 2297, 2299, 2303, 2305, 356 - 359, 806 - 938, 959 - 978, 2180, 2181, 2220 - 2258, 2262 - 2289, 2301, 2307, and 2309 or a modified form thereof.

8. The composition according to any one of the preceding claims, wherein the siRNA molecule comprises A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, M1, N1, O1, P1, Q1, R1, S1, T1, U1, V1, W1, X1, Y1, Z1, A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, M2, N2, O2, P2, Q2, R2, S2, T2, U2, V2, W2, X2, Y2, Z2, A3, B3, C3, D3, E3, F3, G3, H3, I3, J3, K3, L3, M3, N3, O3, P3, Q3, R3, S3, T3, U3, V3, W3, X3, Y3, Z3, A4, B4, C4, D4, E4, F4, G4, H4, I4, J4, K4, L4, M4, N4, O4, P4, Q4, R4, S4, T4, U4, V4, W4, X4, Y4, Z4, A5, B5, C5, D5, E5, F5, G5, H5, I5, J5, K5, L5, M5, N5, O5, P5, Q5, R5, S5, T5, U5, V5, W5, X5, Y5, Z5, A6, B6, C6, D6, E6, F6, G6, H6, I6, J6, K6, L6, M6, N6, O6, P6, Q6, R6, S6, T6, U6, V6, W6, B7, C7, P8, Q8, R8, S8, T8, U8, V8, W8, X8, Y8, Z8, A9, B9, C9, D9, E9, F9, G9, H9, I9, J9, K9, L9, M9, N9, O9, P9, Q9, R9, S9, T9, U9, V9, W9, X9, Y9, Z9, A10, B10, F10, G10, H10, I10, J10, K10, L10, M10, N10, O10, P10, Q10, R10, S10, T10, U10, V10, W10, X10, Y10, Z10, A11, B11, C11, D11, E11, F11, G11, H11, I11, J11, K11, L11, M11, N11, O11, P11, Q11, R11 or a pair of siRNAs (sense strand and antisense strand) as shown in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A or Table 5B.

9. The composition according to claim 8, wherein the composition comprises a pair of siRNAs as shown in Table 3A, Table 3B, Table 4A, Table 4B, Table 5A or Table 5B, which have a linker and / or vinylphosphonate modification as provided herein.

10. The composition according to claim 1, wherein the siRNA molecule has the formula as shown in Formula III: N1N2N3N4N5N6N7N8N9N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 Sense strand (SS) N 21 N 20 N 19 N 18 N 17 N 16 N 15 N 14 N 13 N2N 11 N 10 N9N8N7N6N5N N3 N2N1 antisense strand (AS) (III), wherein N represents each nucleotide as shown in SEQ ID NO: 2110 and SEQ ID NO: 2220, SEQ ID NO: 2113 and SEQ ID NO: 2223, SEQ ID NO: 2111 and SEQ ID NO: 2221, or as shown by the sense and antisense strand pairs (paired siRNAs) provided herein, or comprises modified nucleobases such as those provided herein.

11. The composition according to claim 10, wherein the sense strand comprises 2'-O-methyl modified nucleotides having a phosphorothioate (PS) modified backbone at N1 and N2; 2'-fluoro modified nucleotides at N3, N7, N8, N9, N 12 and N 17 ; and 2'-O-methyl modified nucleotides at N4, N5, N6, N 10 , N 11 , N 13 , N 14 , N 15 , N 16 , N 18 and N 19 .

12. The composition according to claim 10 or 11, wherein the antisense strand comprises a vinyl phosphonate moiety having a phosphorothioate (PS)-modified backbone linked at N1; a 2'-fluoro-modified nucleotide having a PS-modified backbone at N2; 2'-O-methyl-modified nucleotides at N3, N4, N5, N6, N7, N8, N9, N 10 , N 11 , N 12 , N 13 , N 15 , N 16 , N 17 , N 18 and N 19 ; 2'-fluoro-modified nucleotides at N 14 ; and 2'-O-methyl-modified nucleotides having a PS-modified backbone at N 20 and N 21 .

13. The composition according to claim 10, wherein the antisense strand comprises a vinyl phosphonate moiety linked to N1.

14. The composition according to claim 10, wherein the siRNA molecule is conjugated to a linker as shown by the following formula:

15. The composition according to claim 1, wherein the siRNA molecule has the formula as shown by the following formula: wherein F1 is a polypeptide comprising at least one FN3 domain.

16. The composition according to claim 1, the composition further comprising one or more FN3 domains conjugated to the siRNA molecule.

17. The composition according to claim 16, wherein the one or more FN3 domains comprise the FN3 domain conjugated to the siRNA molecule via a cysteine in the FN3 domain.

18. The composition according to claim 16 or 17, wherein the one or more FN3 domains comprise an FN3 domain that binds CD71.

19. The composition according to claim 18, wherein the FN3 domain that binds CD71 comprises an amino acid sequence that is at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of the sequences selected from SEQ ID NO: 570, 672, 1848, 1773, 1849, 1767, 360 - 569, 571 - 644, 663 - 67, 1395 - 1772, 1774 - 1766, 1768 - 1847, and 2310 or is identical thereto.

20. The composition according to claim 18, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 570, SEQ ID NO: 2310, or SEQ ID NO:

672.

21. The composition according to claim 18, wherein the FN3 domain comprises an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 570 or SEQ ID NO: 2310.

22. The composition according to claim 18, wherein the FN3 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 570, provided that the residue at position 54 is cysteine, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2310, provided that the residue at position 53 is cysteine.

23. A pharmaceutical composition, the pharmaceutical composition comprising the composition according to any one of claims 1-22.

24. A kit, the kit comprising the composition according to any one of claims 1-22.

25. A method of treating an immune disease in a subject in need thereof, the method comprising administering to the subject the composition according to any one of claims 1-22.

26. The method according to claim 25, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Sjogren's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis or prevention of solid organ transplant rejection.

27. Use of a composition according to any one of claims 1-22 for the preparation of a pharmaceutical composition or a medicament for treating an immune disease, such as an autoimmune disease.

28. The use according to claim 27, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Sjogren's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis or prevention of solid organ transplant rejection.

29. Use of a composition according to any one of claims 1-22 for treating an immune disease, such as an autoimmune disease.

30. The use according to claim 29, wherein the immune disease is rheumatoid arthritis, Hashimoto's autoimmune thyroiditis, celiac disease, type 1 diabetes, vitiligo, rheumatic fever, pernicious anemia / atrophic gastritis, alopecia areata, immune thrombocytopenic purpura, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, pemphigus, Sjogren's syndrome, inflammatory myositis, lupus nephritis, pemphigus vulgaris, multiple sclerosis or prevention of solid organ transplant rejection.

31. A method of reducing the expression of CD40 in a cell, the method comprising contacting the cell with the composition according to any one of claims 1-22.

32. A method of delivering an siRNA molecule to immune cells of a subject, the method comprising administering to the subject a pharmaceutical composition comprising the composition according to any one of claims 1-22, or the composition comprises an siRNA targeting an immune-specific cellular gene target.

33. The method according to claim 32, wherein the immune cells are B cells, T cells or dendritic cells.

34. The method according to claim 32 or 33, wherein the target gene is CD40.

35. A method of delivering a CD40-targeting siRNA molecule to CD71-positive immune cells of a subject, the method comprising administering to the subject a pharmaceutical composition comprising the composition of any one of claims 1-22, wherein the siRNA molecule downregulates the expression of CD40 in the CD71-positive immune cells.

36. A method of reducing one or more cytokines in a subject, the method comprising administering to the subject the composition of any one of claims 1-22.

37. The method of claim 36, wherein the one or more cytokines are selected from IFN-γ, IL-6, TNF-α, IL-12, IP-10, RANTES, and any combination thereof.

38. A method of reducing or inhibiting the migration of a population of immune cells from blood to tissue, the method comprising contacting the cell population with a composition comprising a CD40-targeting siRNA molecule.

39. The method of claim 38, wherein the composition is the composition of any one of claims 1-22.

40. The method according to claim 38, wherein the population of immune cells comprises cells expressing CD40 (CD40 + ).

41. The method of any one of claims 38-40, wherein the population of immune cells comprises dendritic cells, B cells, or a combination thereof.

Images